Sequences of Brachyspira, immunogenic compounds, methods for preparation and use thereof

ABSTRACT

Novel polynucleotide and amino acids of  Brachyspira hyodysenteriae  are described. These sequences are useful for diagnosis of  B. hyodysenteriae  disease in animals and as a therapeutic treatment or prophylactic treatment of  B. hyodysenteriae  disease in animals. These sequences may also be useful for diagnostic and therapeutic and/or prophylactic treatment of diseases in animals caused by other  Brachyspira  species.

FIELD

This invention relates to novel genes in Brachyspira hyodysenteriae andthe proteins encoded therein. This invention further relates to use ofthese novel genes and proteins for diagnosis of B. hyodysenteriaedisease, vaccines against B. hyodysenteriae and for screening forcompounds that kill B. hyodysenteriae or block the pathogenic effects ofB. hyodysenteriae. These sequences may also be useful for diagnostic andtherapeutic and/or prophylactic treatment of diseases in animals causedby other Brachyspira species, including B. intermedia, B. alvinipulli,B. aalborgi, B. innocens, B. murdochii, and B. pilosicoli.

BACKGROUND

Swine dysentery is a significant endemic disease of pigs in Australiaand worldwide. Swine dysentery is a contagious mucohaemorrhagicdiarrhoeal disease, characterised by extensive inflammation and necrosisof the epithelial surface of the large intestine. Economic losses due toswine dysentery result mainly from growth retardation, costs ofmedication and mortality. The causative agent of swine dysentery wasfirst identified as an anaerobic spirochaete (Treponema hyodysenteriae)in 1971, and was recently reassigned to the genus Brachyspira as B.hyodysenteriae. Where swine dysentery is established in a piggery, thedisease spectrum can vary from being mild, transient or unapparent, tobeing severe and even fatal. Medication strategies on individualpiggeries may mask clinical signs and on some piggeries the disease maygo unnoticed, or may only be suspected. Whether or not obvious diseaseoccurs, B. hyodysenteriae may persist in infected pigs, or in otherreservoir hosts such as rodents, or in the environment. All thesesources pose potential for transmission of the disease to uninfectedherds. Commercial poultry may also be colonized by B. hyodysenteriae,although it is not clear how commonly this occurs under fieldconditions.

Colonisation by B. hyodysenteriae elicits a strong immunologicalresponse against the spirochaete, hence indirect evidence of exposure tothe spirochaete can be obtained by measuring circulating antibody titresin the blood of infected animals. These antibody titres have beenreported to be maintained at low levels, even in animals that haverecovered from swine dysentery. Serological tests for detection ofantibodies therefore have considerable potential for detectingsubclinical infections and recovered carrier pigs that have undetectablenumbers of spirochaetes in their large intestines. These tests would beparticularly valuable in an easy to use kit form, such as anenzyme-linked immunosorbent assay. A variety of techniques have beendeveloped to demonstrate the presence of circulating antibodies againstB. hyodysenteriae, including indirect fluorescent antibody tests,haemagglutination tests, microtitration agglutination tests, complementfixation tests, and ELISA using either lipopolysaccharide or wholesonicated spirochaetes as antigen. All these tests have suffered fromproblems of specificity, as related non-pathogenic intestinalspirochaetes can induce cross-reactive antibodies. These tests areuseful for detecting herds where there is obvious disease and highcirculating antibody titres, but they are problematic for identifyingsub-clinically infected herds and individual infected pigs.Consequently, to date, no completely sensitive and specific assays areavailable for the detection of antibodies against B. hyodysenteriae. Thelack of suitable diagnostic tests has hampered control of swinedysentery.

A number of methods are employed to control swine dysentery, varyingfrom the prophylactic use of antimicrobial agents, to completedestocking of infected herds and prevention of re-entry of infectedcarrier pigs. All these options are expensive and, if they are to befully effective, they require the use of sophisticated diagnostic teststo monitor progress. Currently, detection of swine dysentery in herdswith sub-clinical infections, and individual healthy carrier animals,remains a major problem and is hampering implementation of effectivecontrol measures. A definitive diagnosis of swine dysenterytraditionally has required the isolation and identification of B.hyodysenteriae from the faeces or mucosa of diseased pigs. Majorproblems involved include the slow growth and fastidious nutritionalrequirements of these anaerobic bacteria and confusion due to thepresence of morphologically similar spirochaetes in the normal flora ofthe pig intestine. A significant improvement in the diagnosis ofindividual affected pigs was achieved with the development of polymerasechain reaction (PCR) assays for the detection of spirochaetes fromfaeces. Unfortunately in practical applications the limit of detectionof PCRs rendered it unable to detect carrier animals with subclinicalinfections. As a consequence of these diagnostic problems, there is aclear need to develop a simple and effective diagnostic tool capable ofdetecting B. hyodysenteriae infection at the herd and individual piglevel.

A strong immunological response is induced against the spirochaetefollowing colonization with B. hyodysenteriae, and pigs recovered fromswine dysentery are protected from re-infection. Despite this, attemptsto develop vaccines to control swine dysentery have met with verylimited success, either because they have provided inadequate protectionon a herd basis, or they have been too costly and difficult to produceto make them commercially viable. Bacterin vaccines provide some levelof protection, but they tend to be lipopolysaccharideserogroup-specific, which then requires the use of multivalentbacterins. Furthermore they are difficult and costly to produce on alarge scale because of the fastidious anaerobic growth requirements ofthe spirochaete.

Several attempts have been made to develop attenuated live vaccines forswine dysentery. This approach has the disadvantage that attenuatedstrains show reduced colonisation, and hence cause reduced immunestimulation. There also is reluctance on the part of producers andveterinarians to use live vaccines for swine dysentery because of thepossibility of reversion to virulence, especially as very little isknown about genetic regulation and organization in B. hyodysenteriae.

The use of recombinant subunit vaccines is an attractive alternative,since the products would be well-defined (essential for registrationpurposes), and relatively easy to produce on a large scale. To date thefirst reported use of a recombinant protein from B. hyodysenteriae as avaccine candidate (a 38-kilodalton flagellar protein) failed to preventcolonisation in pigs. This failure is likely to relate specifically tothe particular recombinant protein used, as well as to other moredown-stream issues of delivery systems and routes, dose rates, choice ofadjuvants etc. (Gabe, J D, Chang, R J, Slomiany, R, Andrews, W H andMcCaman, M T (1995) Isolation of extracytoplasmic proteins fromSerpulina hyodysenteriae B204 and molecular cloning of the flaB1 geneencoding a 38-kilodalton flagellar protein. Infection and Immunity63:142-148). The first reported partially protective recombinant B.hyodysenteriae protein used for vaccination was a 29.7 kDa outermembrane lipoprotein (Bhlp29.7, also referred to as BmpB and BlpA) whichhad homology with the methionine-binding lipoproteins of variouspathogenic bacteria. The use of the his-tagged recombinant Bhlp29.7protein for vaccination of pigs, followed by experimental challenge withB. hyodysenteriae, resulted in 17-40% of vaccinated pigs developingdisease compared to 50-70% of the unvaccinated control pigs developingdisease. Since the incidence of disease for the Bhlp29.7 vaccinated pigswas significantly (P=0.047) less than for the control pigs, Bhlp29.7appeared to have potential as a swine dysentery vaccine component (La,T, Phillips, N D, Reichel, M P and Hampson, D J (2004). Protection ofpigs from swine dysentery by vaccination with recombinant BmpB, a 29.7kDa outer-membrane lipoprotein of Brachyspira hyodysenteriae. VeterinaryMicrobiology 102:97-109). A number of other attempts have been made toidentify outer envelop proteins from B. hyodysenteriae that could beused as recombinant vaccine components, but again no successful vaccinehas yet been made. A much more global approach is needed to theidentification of potentially useful immunogenic recombinant proteinsfrom B. hyodysenteriae is needed.

To date, only one study using DNA for vaccination has been reported. Inthis study, the B. hyodysenteriae ftnA gene, encoding a putativeferritin, was cloned into an E. coli plasmid and the plasmid DNA used tocoat gold beads for ballistic vaccination. A murine model for swinedysentery was used to determine the protective nature of vaccinationwith DNA and/or recombinant protein. Vaccination with recombinantprotein induced a good systemic response against ferritin howevervaccination with DNA induced only a detectable systemic response.Vaccination with DNA followed a boost with recombinant protein induced asystemic immune response to ferritin only after boosting with protein.However, none of the vaccination regimes tested was able to provide themice with protection against B. hyodysenteriae colonisation and theassociated lesions. Interestingly, vaccination of the mice with DNAalone resulted in significant exacerbation of disease (Davis, A. J.,Smith, S. C. and Moore, R. J. (2005). The Brachyspira hyodysenteriaeftnA gene: DNA vaccination and real-time PCR quantification of bacteriain a mouse model of disease. Current Microbiology 50: 285-291).

SUMMARY

Inventors have identified novel genes from B. hyodysenteriae and theproteins encoded by those genes. They have further identified that thesenovel genes and proteins can be used for therapeutic and diagnosticpurposes. Moreover, the inventors have identified that these genesand/or the proteins can be used as a vaccine against B. hyodysenteriaeand/or diagnose B. hyodysenteriae infections. Accordingly, in a firstaspect, the present invention provides an isolated polynucleotidecomprising a nucleotide sequence selected from the group consisting ofSEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19. The present inventionalso provides nucleotide sequences that are homologous to SEQ ID NOs: 1,3, 5, 7, 9, 11, 13, 15, 17, and 19, where the homology can be 95%, 90%,85%, 80%, 75% and 70%. This invention also includes a DNA vaccine or DNAimmunogenic composition containing the nucleotide sequence of SEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 and sequences that are 95%,90%, 85%, 80%, 75% and 70% homologous to these sequences. This inventionfurther includes a diagnostic assay containing DNA having the nucleotidesequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19, andsequences that are 95%, 90%, 85%, 80%, 75% and 70% homologous to thesesequences.

In one aspect of the invention, the vaccine composition is a vaccinecomposition comprising at least two polynucleotides encoding moleculesselected from the group consisting of SEQ ID No. 1, 3, 5 and 7 for thetreatment or prevention of swine dysentery associated with Brachyspirahyodysenteriae.

In another aspect of the invention, the vaccine composition is a vaccinecomposition comprising at least three polynucleotides encoding moleculesselected from the group consisting of SEQ ID No. 1, 3, 5 and 7 for thetreatment or prevention of swine dysentery associated with Brachyspirahyodysenteriae.

In yet another aspect of the invention, the vaccine composition is avaccine composition consisting of polynucleotides encoding moleculesselected from the group consisting of: SEQ ID No. 1, 3, 5 and 7 for thetreatment or prevention of swine dysentery associated with Brachyspirahyodysenteriae.

In a further aspect of the invention, the vaccine composition is avaccine composition comprising at least two polynucleotides encodingmolecules selected from the group consisting of SEQ ID No. 9, 11, 13 and15 for the treatment or prevention of swine dysentery associated withBrachyspira hyodysenteriae.

In another aspect of the invention, the vaccine composition is a vaccinecomposition comprising at least three polynucleotides encoding moleculesselected from the group consisting of SEQ ID No. 9, 11, 13 and 15 forthe treatment or prevention of swine dysentery associated withBrachyspira hyodysenteriae.

In yet another aspect of the invention, the vaccine composition is avaccine composition consisting of polynucleotides encoding moleculesselected from the group consisting of: SEQ ID No. 9, 11, 13 and 15 forthe treatment or prevention of swine dysentery associated withBrachyspira hyodysenteriae.

In a further aspect of the invention, the vaccine composition is avaccine composition comprising at least two polynucleotides encodingmolecules selected from the group consisting of SEQ ID No. 17, and 19for the treatment or prevention of swine dysentery associated withBrachyspira hyodysenteriae.

In another aspect of the invention, the vaccine composition is a vaccinecomposition comprising:

(i) at least one polynucleotide encoding a molecule selected from thegroup of: SEQ ID No. 1, 3, 5 and 7; and/or

(ii) at least one polynucleotide encoding a molecule selected from thegroup of: SEQ ID No. 9, 11, 13 and 15 and/or

(iii) at least one polynucleotide encoding a molecule selected from thegroup of: SEQ ID No. 17 and for the treatment or prevention of swinedysentery associated with Brachyspira.

The present invention also provides plasmids containing DNA having thesequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19;prokaryotic and/or eukaryotic expression vectors containing DNA havingthe sequence of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, and 19; and acell containing the plasmids which contain DNA having the sequence ofSEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19.

In a second aspect, the present invention provides an isolatedpolypeptide comprising the amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20. Thepresent invention also provides novel B. hyodysenteriae proteins havingthe amino acid sequence contained in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,16, 18, and 20. This invention also provides proteins that are 95%, 90%,85%, 80%, 75% and 70% homologous to the sequences contained in SEQ IDNOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20. The present invention alsoprovides a vaccine or immunogenic composition to contain the proteinshaving the amino acid sequence contained in SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 16, 18, and 20 or amino acid sequences that are 95%, 90%, 85%,80%, 75% and 70% homologous to the sequences contained in SEQ ID NOs: 2,4, 6, 8, 10, 12, 14, 16, 18, and 20.

In one aspect of the invention, the vaccine composition is a vaccinecomposition comprising at least two polypeptides selected from the groupconsisting of SEQ ID No. 2, 4, 6 and 8 for the treatment or preventionof swine dysentery associated with Brachyspira hyodysenteriae.

In another aspect of the invention, the vaccine composition is a vaccinecomposition comprising at least three polypeptides selected from thegroup consisting of SEQ ID No. 2, 4, 6 and 8 for the treatment orprevention of swine dysentery associated with Brachyspirahyodysenteriae.

In yet another aspect of the invention, the vaccine composition is avaccine composition consisting of polypeptides SEQ ID No. 2, 4, 6 and 8for the treatment or prevention of swine dysentery associated withBrachyspira hyodysenteriae.

In one aspect of the invention, the vaccine composition is a vaccinecomposition comprising at least two polypeptides selected from the groupconsisting of SEQ ID No. 10, 12, 14 and 16 for the treatment orprevention of swine dysentery associated with Brachyspirahyodysenteriae.

In another aspect of the invention, the vaccine composition is a vaccinecomposition comprising at least three polypeptides selected from thegroup consisting of SEQ ID No. 10, 12, 14 and 16 for the treatment orprevention of swine dysentery associated with Brachyspirahyodysenteriae.

In yet another aspect of the invention, the vaccine composition is avaccine composition consisting of polypeptides from the group consistingof: SEQ ID No. 10, 12, 14 and 16 for the treatment or prevention ofswine dysentery associated with Brachyspira hyodysenteriae.

In one aspect of the invention, the vaccine composition is a vaccinecomposition comprising at least two polypeptides selected from the groupconsisting of SEQ ID No. 18 and 20 for the treatment or prevention ofswine dysentery associated with Brachyspira hyodysenteriae.

In a further aspect of the invention, the vaccine composition is avaccine composition comprising:

(i) at least one polypeptide selected from the group of: SEQ ID No. 2,4, 6, or 8 and/or

(ii) at least one polypeptide selected from the group of: SEQ ID No. 10,12, 14, or 16 and/or

(iii) at least one selected from the group of: SEQ ID No. 18 and 20 forthe treatment or prevention of swine dysentery associated withBrachyspira hyodysenteriae.

In a third aspect, the present invention provides a method of diagnosingBrachyspira infection comprising: (a) providing a sample from an animalsuspected of being infected with Brachyspira; (b) contacting the samplewith one or more polypeptides comprising the amino acid sequenceselected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,16, 18, and 20; (c) incubating the sample and polypeptide underconditions which allow for the formation of antibody-antigen complexes;and (d) determining whether an antibody-antigen complex with one or morepolypeptides is formed, wherein the formation of an antibody-antigencomplex indicates the animal is infected with Brachyspira.

In a fourth aspect, the present invention provides a kit for diagnosingBrachyspira infection comprising one or more polypeptides comprising theamino acid sequence selected from the group consisting of SEQ ID NO: 2,4, 6, 8, 10, 12, 14, 16, 18, and 20.

It is a further aspect of this invention to have a diagnostic kitcontaining one or more proteins having a sequence contained in SEQ IDNOs: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 or that are 95%, 90%, 85%,80%, 75% and 70% homologous to the sequences contained in SEQ ID NOs: 2,4, 6, 8, 10, 12, 14, 16, 18, and 20.

It is another aspect of this invention to have nucleotide sequenceswhich encode the proteins having the amino acid sequence contained inSEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20. The invention alsocovers plasmids, eukaryotic and prokaryotic expression vectors, and DNAvaccines which contain DNA having a sequence which encodes a proteinhaving the amino acid sequence contained in SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 16, 18, and 20. Cells which contain these plasmids andexpression vectors are included in this invention.

This invention includes monoclonal antibodies that bind to proteinshaving an amino acid sequence contained in SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 16, 18, and 20 or bind to proteins that are 95%, 90%, 85%, 80%,75% and 70% homologous to the sequences contained in SEQ ID NOs: 2, 4,6, 8, 10, 12, 14, 16, 18, and 20. Diagnostic kits containing themonoclonal antibodies that bind to proteins having an amino acidsequence contained in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20or bind to proteins that are 95%, 90%, 85%, 80%, 75% and 70% homologousto the sequences contained in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16,18, and 20 are included in this invention. These diagnostic kits candetect the presence of B. hyodysenteriae in an animal. The animal ispreferably a mammal or a bird; more preferably, chicken, goose, duck,turkey, parakeet, dog, cat, hamster, gerbil, rabbit, ferret, horse, cow,sheep, pig, monkey, and human.

The invention also contemplates the method of preventing or treating aninfection of B. hyodysenteriae in an animal by administering to ananimal a DNA vaccine containing one or more nucleotide sequences listedin SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 or sequences thatare 95%, 90%, 85%, 80%, 75% and 70% homologous to these sequences. Thisinvention also covers a method of preventing or treating an infection ofB. hyodysenteriae in an animal by administering to an animal a vaccinecontaining one or more proteins having the amino acid sequencecontaining in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 orsequences that are 95%, 90%, 85%, 80%, 75% and 70% homologous to thesesequences.

The invention also contemplates the method of generating an immuneresponse in an animal by administering to an animal an immunogeniccomposition containing one or more nucleotide sequences listed in SEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 or sequences that are 95%,90%, 85%, 80%, 75% and 70% homologous to these sequences. This inventionalso covers a method of generating an immune response in an animal byadministering to an animal an immunogenic composition containing one ormore proteins having the amino acid sequence containing in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 or sequences that are 95%, 90%,85%, 80%, 75% and 70% homologous to these sequences.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified methods and may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting which will be limited only by the appendedclaims.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.However, publications mentioned herein are cited for the purpose ofdescribing and disclosing the protocols, reagents and vectors which arereported in the publications and which might be used in connection withthe invention. Nothing herein is to be construed as an admission thatthe invention is not entitled to antedate such disclosure by virtue ofprior invention.

Furthermore, the practice of the present invention employs, unlessotherwise indicated, conventional immunological and molecular biologicaltechniques and pharmacology within the skill of the art. Such techniquesare well known to the skilled worker, and are explained fully in theliterature. See, eg., Coligan, Dunn, Ploegh, Speicher and Wingfield“Current protocols in Protein Science” (1999) Volume I and II (JohnWiley & Sons Inc.); Sambrook et al., “Molecular Cloning: A LaboratoryManual” (1989), 2^(nd) Edition (Cold Spring Harbor Laboratory press);and Prescott, Harley and Klein “Microbiology” (1999), 4^(th) Edition(WBC McGraw-Hill).

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “agene” includes a plurality of such genes, and a reference to “an animal”is a reference to one or more animals, and so forth. Unless definedotherwise, all technical and scientific terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art towhich this invention belongs. Although any materials and methods similaror equivalent to those described herein can be used to practice or testthe present invention, the preferred materials and methods are nowdescribed.

In the broadest aspect of the invention there is provided a novel B.hyodysenteriae polynucleotide having the nucleotide sequence containedin SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23 or an aminoacid sequence (polypeptide) encoded by these polynucleotides.

The term “amino acid” is intended to embrace all molecules, whethernatural or synthetic, which include both an amino functionality and anacid functionality and capable of being included in a polymer ofnaturally-occurring amino acids. Exemplary amino acids includenaturally-occurring amino acids; analogs, derivatives and congenersthereof; amino acid analogs having variant side chains; and allstereoisomers of any of any of the foregoing.

An animal can be a mammal or a bird that can be infected withBrachyspira, especially B. hyodysenteriae. Examples of mammals includedog, cat, hamster, gerbil, rabbit, ferret, horse, cow, sheep, pig,monkey, and human. Examples of birds include chicken, goose, duck,turkey, and parakeet. It is appreciated by those skilled in the art thatcertain Brachyspira species are capable of infecting a broad host range(see, for example, Hampson et al., 2006, Emerging Infectious diseases,Vol. 12(5), p 869-870, incorporated herein in its entirety byreference). Accordingly the term “animal” as used herein encompasses arange of animals, but especially pigs and poultry.

The term “conserved residue” refers to an amino acid that is a member ofa group of amino acids having certain common properties. The term“conservative amino acid substitution” refers to the substitution(conceptually or otherwise) of an amino acid from one such group with adifferent amino acid from the same group. A functional way to definecommon properties between individual amino acids is to analyze thenormalized frequencies of amino acid changes between correspondingproteins of homologous organisms (Schulz, G. E. and R. H. Schinner.,Principles of Protein Structure, Springer-Verlag). According to suchanalyses, groups of amino acids may be defined where amino acids withina group exchange preferentially with each other, and therefore resembleeach other most in their impact on the overall protein structure(Schulz, G. E. and R. H. Schirmer, Principles of Protein Structure,Springer-Verlag). Examples of amino acid groups defined in this mannerinclude: (i) a positively-charged group containing Lys, Arg and His,(ii) a negatively-charged group containing Glu and Asp, (iii) anaromatic group containing Phe, Tyr and Trp, (iv) a nitrogen ring groupcontaining His and Trp, (v) a large aliphatic nonpolar group containingVal, Leu and De, (vi) a slightly-polar group containing Met and Cys,(vii) a small-residue group containing Ser, Thr, Asp, Asn, Gly, Ala,Glu, Gln and Pro, (viii) an aliphatic group containing Val, Leu, Ile,Met and Cys, and (ix) a small, hydroxyl group containing Ser and Thr.

A “fusion protein” or “fusion polypeptide” refers to a chimeric proteinas that term is known in the art and may be constructed using methodsknown in the art. In many examples of fusion proteins, there are twodifferent polypeptide sequences, and in certain cases, there may bemore. The polynucleotide sequences encoding the fusion protein may beoperably linked in frame so that the fusion protein may be translatedcorrectly. A fusion protein may include polypeptide sequences from thesame species or from different species. In various embodiments, thefusion polypeptide may contain one or more amino acid sequences linkedto a first polypeptide. In the case where more than one amino acidsequence is fused to a first polypeptide, the fusion sequences may bemultiple copies of the same sequence, or alternatively, may be differentamino acid sequences. The fusion polypeptides may be fused to theN-terminus, the C-terminus, or the N- and C-terminus of the firstpolypeptide. Exemplary fusion proteins include polypeptides containing aglutathione S-transferase tag (GST-tag), histidine tag (His-tag), animmunoglobulin domain or an immunoglobulin binding domain.

The term “isolated polypeptide” refers to a polypeptide, in certainembodiments prepared from recombinant DNA or RNA, or of synthetic originor natural origin, or some combination thereof, which (1) is notassociated with proteins that it is normally found with in nature, (2)is separated from the cell in which it normally occurs, (3) is free ofother proteins from the same cellular source, (4) is expressed by a cellfrom a different species, or (5) does not occur in nature. It ispossible for an isolated polypeptide to exist, but not qualify as apurified polypeptide.

The term “isolated nucleic acid” and “isolated polynucleotide” refers toa polynucleotide whether genomic DNA, cDNA, mRNA, tRNA, rRNA, iRNA, or apolynucleotide obtained from a cellular organelle (such as mitochondriaand chloroplast), or whether from synthetic origin, which (1) is notassociated with the cell in which the “isolated nucleic acid” is foundin nature, or (2) is operably linked to a polynucleotide to which it isnot linked in nature. It is possible for an isolated polynucleotide toexist, but not qualify as a purified polynucleotide.

The term “nucleic acid” and “polynucleotide” refers to a polymeric formof nucleotides, either ribonucleotides or deoxyribonucleotides or amodified form of either type of nucleotide. The terms should also beunderstood to include, as equivalents, analogs of either RNA or DNA madefrom nucleotide analogs, and, as applicable to the embodiment beingdescribed, single-stranded (such as sense or antisense) anddouble-stranded polynucleotides.

The term “nucleic acid of the invention” and “polynucleotide of theinvention” refers to a nucleic acid encoding a polypeptide of theinvention. A polynucleotide of the invention may comprise all, or aportion of, a subject nucleic acid sequence; a nucleotide sequence atleast 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to asubject nucleic acid sequence; a nucleotide sequence that hybridizesunder stringent conditions to a subject nucleic acid sequence;nucleotide sequences encoding polypeptides that are functionallyequivalent to polypeptides of the invention; nucleotide sequencesencoding polypeptides at least about 60%, 70%, 80%, 85%, 90%, 95%, 98%,99% homologous or identical with a subject amino acid sequence;nucleotide sequences encoding polypeptides having an activity of apolypeptide of the invention and having at least about 60%, 70%, 80%,85%, 90%, 95%, 98%, 99% or more homology or identity with a subjectamino acid sequence; nucleotide sequences that differ by 1 to about 2,3, 5, 7, 10, 15, 20, 30, 50, 75 or more nucleotide substitutions,additions or deletions, such as allelic variants, of a subject nucleicacid sequence; nucleic acids derived from and evolutionarily related toa subject nucleic acid sequence; and complements of, and nucleotidesequences resulting from the degeneracy of the genetic code, for all ofthe foregoing and other nucleic acids of the invention. Nucleic acids ofthe invention also include homologs, e.g., orthologs and paralogs, of asubject nucleic acid sequence and also variants of a subject nucleicacid sequence which have been codon optimized for expression in aparticular organism (e.g., host cell).

The term “operably linked”, when describing the relationship between twonucleic acid regions, refers to a juxtaposition wherein the regions arein a relationship permitting them to function in their intended manner.For example, a control sequence “operably linked” to a coding sequenceis ligated in such a way that expression of the coding sequence isachieved under conditions compatible with the control sequences, such aswhen the appropriate molecules (e.g., inducers and polymerases) arebound to the control or regulatory sequence(s).

The term “polypeptide”, and the terms “protein” and “peptide” which areused interchangeably herein, refers to a polymer of amino acids.Exemplary polypeptides include gene products, naturally-occurringproteins, homologs, orthologs, paralogs, fragments, and otherequivalents, variants and analogs of the foregoing.

The terms “polypeptide fragment” or “fragment”, when used in referenceto a reference polypeptide, refers to a polypeptide in which amino acidresidues are deleted as compared to the reference polypeptide itself,but where the remaining amino acid sequence is usually identical to thecorresponding positions in the reference polypeptide. Such deletions mayoccur at the amino-terminus or carboxy-terminus of the referencepolypeptide, or alternatively both. Fragments typically are at least 5,6, 8 or 10 amino acids long, at least 14 amino acids long, at least 20,30, 40 or 50 amino acids long, at least 75 amino acids long, or at least100, 150, 200, 300, 500 or more amino acids long. A fragment can retainone or more of the biological activities of the reference polypeptide.In certain embodiments, a fragment may comprise a domain having thedesired biological activity, and optionally additional amino acids onone or both sides of the domain, which additional amino acids may numberfrom 5, 10, 15, 20, 30, 40, 50, or up to 100 or more residues. Further,fragments can include a sub-fragment of a specific region, whichsub-fragment retains a function of the region from which it is derived.In another embodiment, a fragment may have immunogenic properties.

The term “polypeptide of the invention” refers to a polypeptidecontaining a subject amino acid sequence, or an equivalent or fragmentthereof. Polypeptides of the invention include polypeptides containingall or a portion of a subject amino acid sequence; a subject amino acidsequence with 1 to about 2, 3, 5, 7, 10, 15, 20, 30, 50, 75 or moreconservative amino acid substitutions; an amino acid sequence that is atleast 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to asubject amino acid sequence; and functional fragments thereof.Polypeptides of the invention also include homologs, e.g., orthologs andparalogs, of a subject amino acid sequence.

It is also possible to modify the structure of the polypeptides of theinvention for such purposes as enhancing therapeutic or prophylacticefficacy, or stability (e.g., ex vivo shelf life, resistance toproteolytic degradation in vivo, etc.). Such modified polypeptides, whendesigned to retain at least one activity of the naturally-occurring formof the protein, are considered “functional equivalents” of thepolypeptides described in more detail herein. Such modified polypeptidesmay be produced, for instance, by amino acid substitution, deletion, oraddition, which substitutions may consist in whole or part byconservative amino acid substitutions.

For instance, it is reasonable to expect that an isolated conservativeamino acid substitution, such as replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, a threonine with aserine, will not have a major affect on the biological activity of theresulting molecule. Whether a change in the amino acid sequence of apolypeptide results in a functional homolog may be readily determined byassessing the ability of the variant polypeptide to produce a responsesimilar to that of the wild-type protein. Polypeptides in which morethan one replacement has taken place may readily be tested in the samemanner.

The term “purified” refers to an object species that is the predominantspecies present (i.e., on a molar basis it is more abundant than anyother individual species in the composition). A “purified fraction” is acomposition wherein the object species is at least about 50 percent (ona molar basis) of all species present. In making the determination ofthe purity or a species in solution or dispersion, the solvent or matrixin which the species is dissolved or dispersed is usually not includedin such determination; instead, only the species (including the one ofinterest) dissolved or dispersed are taken into account. Generally, apurified composition will have one species that is more than about 80%of all species present in the composition, more than about 85%, 90%,95%, 99% or more of all species present. The object species may bepurified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition is essentially a single species. A skilled artisan maypurify a polypeptide of the invention using standard techniques forprotein purification in light of the teachings herein. Purity of apolypeptide may be determined by a number of methods known to those ofskill in the art, including for example, amino-terminal amino acidsequence analysis, gel electrophoresis, mass-spectrometry analysis andthe methods described herein.

The terms “recombinant protein” or “recombinant polypeptide” refer to apolypeptide which is produced by recombinant DNA techniques. An exampleof such techniques includes the case when DNA encoding the expressedprotein is inserted into a suitable expression vector which is in turnused to transform a host cell to produce the protein or polypeptideencoded by the DNA.

The term “regulatory sequence” is a generic term used throughout thespecification to refer to polynucleotide sequences, such as initiationsignals, enhancers, regulators and promoters, that are necessary ordesirable to affect the expression of coding and non-coding sequences towhich they are operably linked. Exemplary regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology,Academic Press, San Diego, Calif. (1990), and include, for example, theearly and late promoters of SV40, adenovirus or cytomegalovirusimmediate early promoter, the lac system, the trp system, the TAC or TRCsystem, T7 promoter whose expression is directed by T7 RNA polymerase,the major operator and promoter regions of phage lambda, the controlregions for fd coat protein, the promoter for 3-phosphoglycerate kinaseor other glycolytic enzymes, the promoters of acid phosphatase (e.g.,Pho5), the promoters of the yeast α-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. The nature and use of suchcontrol sequences may differ depending upon the host organism. Inprokaryotes, such regulatory sequences generally include promoter,ribosomal binding site, and transcription termination sequences. Theterm “regulatory sequence” is intended to include, at a minimum,components whose presence may influence expression, and may also includeadditional components whose presence is advantageous, for example,leader sequences and fusion partner sequences. In certain embodiments,transcription of a polynucleotide sequence is under the control of apromoter sequence (or other regulatory sequence) which controls theexpression of the polynucleotide in a cell-type in which expression isintended. It will also be understood that the polynucleotide can beunder the control of regulatory sequences which are the same ordifferent from those sequences which control expression of thenaturally-occurring form of the polynucleotide.

The term “sequence homology” refers to the proportion of base matchesbetween two nucleic acid sequences or the proportion of amino acidmatches between two amino acid sequences. When sequence homology isexpressed as a percentage, e.g., 50%, the percentage denotes theproportion of matches over the length of sequence from a desiredsequence that is compared to some other sequence. Gaps (in either of thetwo sequences) are permitted to maximize matching; gap lengths of 15bases or less are usually used, 6 bases or less are used morefrequently, with 2 bases or less used even more frequently. The term“sequence identity” means that sequences are identical (i.e., on anucleotide-by-nucleotide basis for nucleic acids or amino acid-by-aminoacid basis for polypeptides) over a window of comparison. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the comparison window, determining thenumber of positions at which the identical amino acids or nucleotidesoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the comparison window, and multiplying the result by 100 toyield the percentage of sequence identity. Methods to calculate sequenceidentity are known to those of skill in the art and described in furtherdetail below.

The term “soluble” as used herein with reference to a polypeptide of theinvention or other protein, means that upon expression in cell culture,at least some portion of the polypeptide or protein expressed remains inthe cytoplasmic fraction of the cell and does not fractionate with thecellular debris upon lysis and centrifugation of the lysate. Solubilityof a polypeptide may be increased by a variety of art recognizedmethods, including fusion to a heterologous amino acid sequence,deletion of amino acid residues, amino acid substitution (e.g.,enriching the sequence with amino acid residues having hydrophilic sidechains), and chemical modification (e.g., addition of hydrophilicgroups).

The solubility of polypeptides may be measured using a variety of artrecognized techniques, including, dynamic light scattering to determineaggregation state, UV absorption, centrifugation to separate aggregatedfrom non-aggregated material, and SDS gel electrophoresis (e.g., theamount of protein in the soluble fraction is compared to the amount ofprotein in the soluble and insoluble fractions combined). When expressedin a host cell, the polypeptides of the invention may be at least about1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more soluble,e.g., at least about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or more of the total amount of protein expressed in the cell isfound in the cytoplasmic fraction. In certain embodiments, a one literculture of cells expressing a polypeptide of the invention will produceat least about 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 milligrams ofmore of soluble protein. In an exemplary embodiment, a polypeptide ofthe invention is at least about 10% soluble and will produce at leastabout 1 milligram of protein from a one liter cell culture.

The term “specifically hybridizes” refers to detectable and specificnucleic acid binding. Polynucleotides, oligonucleotides and nucleicacids of the invention selectively hybridize to nucleic acid strandsunder hybridization and wash conditions that minimize appreciableamounts of detectable binding to nonspecific nucleic acids. Stringentconditions may be used to achieve selective hybridization conditions asknown in the art and discussed herein. Generally, the nucleic acidsequence homology between the polynucleotides, oligonucleotides, andnucleic acids of the invention and a nucleic acid sequence of interestwill be at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%,or more. In certain instances, hybridization and washing conditions areperformed under stringent conditions according to conventionalhybridization procedures and as described further herein.

The terms “stringent conditions” or “stringent hybridization conditions”refer to conditions which promote specific hybridization between twocomplementary polynucleotide strands so as to form a duplex. Stringentconditions may be selected to be about 5° C. lower than the thermalmelting point (Tm) for a given polynucleotide duplex at a defined ionicstrength and pH. The length of the complementary polynucleotide strandsand their GC content will determine the Tm of the duplex, and thus thehybridization conditions necessary for obtaining a desired specificityof hybridization. The Tm is the temperature (under defined ionicstrength and pH) at which 50% of a polynucleotide sequence hybridizes toa perfectly matched complementary strand. In certain cases it may bedesirable to increase the stringency of the hybridization conditions tobe about equal to the Tm for a particular duplex.

A variety of techniques for estimating the Tm are available. Typically,G-C base pairs in a duplex are estimated to contribute about 3° C. tothe Tm, while A-T base pairs are estimated to contribute about 2° C., upto a theoretical maximum of about 80-100° C.

However, more sophisticated models of Tm are available in which G-Cstacking interactions, solvent effects, the desired assay temperatureand the like are taken into account. For example, probes can be designedto have a dissociation temperature (Td) of approximately 60° C., usingthe formula: Td=(((3×#GC)+(2×NAT))×37)−562)/#bp)−5; where #GC, #AT, and#bp are the number of guanine-cytosine base pairs, the number ofadenine-thymine base pairs, and the number of total base pairs,respectively, involved in the formation of the duplex.

Hybridization may be carried out in 5×SSC, 4×SSC, 3×SSC, 2×SSC, 1×SSC or0.2×SSC for at least about 1 hour, 2 hours, 5 hours, 12 hours, or 24hours. The temperature of the hybridization may be increased to adjustthe stringency of the reaction, for example, from about 25° C. (roomtemperature), to about 45° C., 50° C., 55° C., 60° C., or 65° C. Thehybridization reaction may also include another agent affecting thestringency, for example, hybridization conducted in the presence of 50%formamide increases the stringency of hybridization at a definedtemperature.

The hybridization reaction may be followed by a single wash step, or twoor more wash steps, which may be at the same or a different salinity andtemperature. For example, the temperature of the wash may be increasedto adjust the stringency from about 25° C. (room temperature), to about45° C., 50° C., 55° C., 60° C., 65° C., or higher. The wash step may beconducted in the presence of a detergent, e.g., 0.1 or 0.2% SDS. Forexample, hybridization may be followed by two wash steps at 65° C. eachfor about 20 minutes in 2×SSC, 0.1% SDS, and optionally two additionalwash steps at 65° C. each for about 20 minutes in 0.2×SSC, 0.1% SDS.

Exemplary stringent hybridization conditions include overnighthybridization at 65° C. in a solution containing 50% formamide,10×Denhardt (0.2% Ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovine serumalbumin) and 200 μg/ml of denatured carrier DNA, e.g., sheared salmonsperm DNA, followed by two wash steps at 65° C. each for about 20minutes in 2×SSC, 0.1% SDS, and two wash steps at 65° C. each for about20 minutes in 0.2×SSC, 0.1% SDS.

Hybridization may consist of hybridizing two nucleic acids in solution,or a nucleic acid in solution to a nucleic acid attached to a solidsupport, e.g., a filter. When one nucleic acid is on a solid support, aprehybridization step may be conducted prior to hybridization.Prehybridization may be carried out for at least about 1 hour, 3 hoursor 10 hours in the same solution and at the same temperature as thehybridization solution (without the complementary polynucleotidestrand).

Appropriate stringency conditions are known to those skilled in the artor may be determined experimentally by the skilled artisan. See, forexample, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-12.3.6; Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, N.Y.; S. Agrawal (ed.)Methods in Molecular Biology, volume 20; Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization WithNucleic Acid Probes, e.g., part I chapter 2 “Overview of principles ofhybridization and the strategy of nucleic acid probe assays”, Elsevier,New York; and Tibanyenda, N. et al., Eur. J. Biochem. 139:19 (1984) andEbel, S. et al., Biochem. 31:12083 (1992).

The term “vector” refers to a nucleic acid capable of transportinganother nucleic acid to which it has been linked. One type of vectorwhich may be used in accord with the invention is an episome, i.e., anucleic acid capable of extra-chromosomal replication. Other vectorsinclude those capable of autonomous replication and expression ofnucleic acids to which they are linked. Vectors capable of directing theexpression of genes to which they are operatively linked are referred toherein as “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of“plasmids” which refer to circular double stranded DNA molecules which,in their vector form are not bound to the chromosome. In the presentspecification, “plasmid” and “vector” are used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors whichserve equivalent functions and which become known in the artsubsequently hereto.

The nucleic acids of the invention may be used as diagnostic reagents todetect the presence or absence of the target DNA or RNA sequences towhich they specifically bind, such as for determining the level ofexpression of a nucleic acid of the invention. In one aspect, thepresent invention contemplates a method for detecting the presence of anucleic acid of the invention or a portion thereof in a sample, themethod of the steps of: (a) providing an oligonucleotide at least eightnucleotides in length, the oligonucleotide being complementary to aportion of a nucleic acid of the invention; (b) contacting theoligonucleotide with a sample containing at least one nucleic acid underconditions that permit hybridization of the oligonucleotide with anucleic acid of the invention or a portion thereof; and (c) detectinghybridization of the oligonucleotide to a nucleic acid in the sample,thereby detecting the presence of a nucleic acid of the invention or aportion thereof in the sample. In another aspect, the present inventioncontemplates a method for detecting the presence of a nucleic acid ofthe invention or a portion thereof in a sample, by (a) providing a pairof single stranded oligonucleotides, each of which is at least eightnucleotides in length, complementary to sequences of a nucleic acid ofthe invention, and wherein the sequences to which the oligonucleotidesare complementary are at least ten nucleotides apart; and (b) contactingthe oligonucleotides with a sample containing at least one nucleic acidunder hybridization conditions; (c) amplifying the nucleotide sequencebetween the two oligonucleotide primers; and (d) detecting the presenceof the amplified sequence, thereby detecting the presence of a nucleicacid of the invention or a portion thereof in the sample.

In another aspect of the invention, the polynucleotide of the inventionis provided in an expression vector containing a nucleotide sequenceencoding a polypeptide of the invention and operably linked to at leastone regulatory sequence. It should be understood that the design of theexpression vector may depend on such factors as the choice of the hostcell to be transformed and/or the type of protein desired to beexpressed. The vector's copy number, the ability to control that copynumber and the expression of any other protein encoded by the vector,such as antibiotic markers, should be considered.

An expression vector containing the polynucleotide of the invention canthen be used as a pharmaceutical agent to treat an animal infected withB. hyodysenteriae or as a vaccine (also a pharmaceutical agent) toprevent an animal from being infected with B. hyodysenteriae, or toreduce the symptoms and course of the disease if the animal does becomeinfected. One manner of using an expression vector as a pharmaceuticalagent is to administer a nucleic acid vaccine to the animal at risk ofbeing infected or to the animal after being infected. Nucleic acidvaccine technology is well-described in the art. Some descriptions canbe found in U.S. Pat. No. 6,562,376 (Hooper et al.); U.S. Pat. No.5,589,466 (Felgner, et al.); U.S. Pat. No. 6,673,776 (Felgner, et al.);and U.S. Pat. No. 6,710,035 (Felgner, et al.). Nucleic acid vaccines canbe injected into muscle or intradermally, can be electroporated into theanimal (see WO 01/23537, King et al.; and WO 01/68889, Malone et al.),via lipid compositions (see U.S. Pat. No. 5,703,055, Felgner, et al), orother mechanisms known in the art field.

Expression vectors can also be transfected into bacteria which can beadministered to the target animal to induce an immune response to theprotein encoded by the nucleotides of this invention contained on theexpression vector. The expression vector can contain eukaryoticexpression sequences such that the nucleotides of this invention aretranscribed and translated in the host animal. Alternatively, theexpression vector can be transcribed in the bacteria and then translatedin the host animal. The bacteria used as a carrier of the expressionvector should be attenuated but still invasive. One can use Shigellaspp., Salmonella spp., Escherichia spp., and Aeromonas spp., just toname a few, that have been attenuated but still invasive. Examples ofthese methods can be found in U.S. Pat. No. 5,824,538 (Branstrom et al);U.S. Pat. No. 5,877,159 (Powell, et al.); U.S. Pat. No. 6,150,170(Powell, et al.); U.S. Pat. No. 6,500,419 (Hone, et al.); and U.S. Pat.No. 6,682,729 (Powell, et al.).

Alternatively, the polynucleotides of this invention can be placed incertain viruses which act a vector. Viral vectors can either express theproteins of this invention on the surface of the virus, or carrypolynucleotides of this invention into an animal cell where thepolynucleotide is transcribed and translated into a protein. The animalinfected with the viral vectors can develop an immune response to theproteins encoded by the polynucleotides of this invention. Thereby onecan alleviate or prevent an infection by B. hyodysenteriae in the animalwhich received the viral vectors. Examples of viral vectors can be foundU.S. Pat. No. 5,283,191 (Morgan et al.); U.S. Pat. No. 5,554,525(Sondermeijer et al) and U.S. Pat. No. 5,712,118 (Murphy).

The polynucleotide of the invention may be used to cause expression andover-expression of a polypeptide of the invention in cells propagated inculture, e.g. to produce proteins or polypeptides, including fusionproteins or polypeptides.

This invention pertains to a host cell transfected with a recombinantgene in order to express a polypeptide of the invention. The host cellmay be any prokaryotic or eukaryotic cell. For example, a polypeptide ofthe invention may be expressed in bacterial cells, such as E. coli,insect cells (baculovirus), yeast, plant, or mammalian cells. In thoseinstances when the host cell is human, it may or may not be in a livesubject. Other suitable host cells are known to those skilled in theart. Additionally, the host cell may be supplemented with tRNA moleculesnot typically found in the host so as to optimize expression of thepolypeptide. Alternatively, the nucleotide sequence may be altered tooptimize expression in the host cell, yet the protein produced wouldhave high homology to the originally encoded protein. Other methodssuitable for maximizing expression of the polypeptide will be known tothose in the art.

The present invention further pertains to methods of producing thepolypeptides of the invention. For example, a host cell transfected withan expression vector encoding a polypeptide of the invention may becultured under appropriate conditions to allow expression of thepolypeptide to occur. The polypeptide may be secreted and isolated froma mixture of cells and medium containing the polypeptide. Alternatively,the polypeptide may be retained cytoplasmically and the cells harvested,lysed and the protein isolated.

A cell culture includes host cells, media and other byproducts. Suitablemedia for cell culture are well known in the art. The polypeptide may beisolated from cell culture medium, host cells, or both using techniquesknown in the art for purifying proteins, including ion-exchangechromatography, gel filtration chromatography, ultrafiltration,electrophoresis, and immunoaffinity purification with antibodiesspecific for particular epitopes of a polypeptide of the invention.

Thus, a nucleotide sequence encoding all or a selected portion ofpolypeptide of the invention, may be used to produce a recombinant formof the protein via microbial or eukaryotic cellular processes. Ligatingthe sequence into a polynucleotide construct, such as an expressionvector, and transforming or transfecting into hosts, either eukaryotic(yeast, avian, insect or mammalian) or prokaryotic (bacterial cells),are standard procedures. Similar procedures, or modifications thereof,may be employed to prepare recombinant polypeptides of the invention bymicrobial means or tissue-culture technology.

Suitable vectors for the expression of a polypeptide of the inventioninclude plasmids of the types: pTrcHis-derived plasmids, pET-derivedplasmids, pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derivedplasmids, pBTac-derived plasmids and pUC-derived plasmids for expressionin prokaryotic cells, such as E. coli. The various methods employed inthe preparation of the plasmids and transformation of host organisms arewell known in the art. For other suitable expression systems for bothprokaryotic and eukaryotic cells, as well as general recombinantprocedures, see Molecular Cloning, A Laboratory Manual, 2nd Ed., ed. bySambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press,1989) Chapters 16 and 17.

Coding sequences for a polypeptide of interest may be incorporated as apart of a fusion gene including a nucleotide sequence encoding adifferent polypeptide. The present invention contemplates an isolatedpolynucleotide containing a nucleic acid of the invention and at leastone heterologous sequence encoding a heterologous peptide linked inframe to the nucleotide sequence of the nucleic acid of the invention soas to encode a fusion protein containing the heterologous polypeptide.The heterologous polypeptide may be fused to (a) the C-terminus of thepolypeptide of the invention, (b) the N-terminus of the polypeptide ofthe invention, or (c) the C-terminus and the N-terminus of thepolypeptide of the invention. In certain instances, the heterologoussequence encodes a polypeptide permitting the detection, isolation,solubilization and/or stabilization of the polypeptide to which it isfused. In still other embodiments, the heterologous sequence encodes apolypeptide such as a poly His tag, myc, HA, GST, protein A, protein G,calmodulin-binding peptide, thioredoxin, maltose-binding protein, polyarginine, poly His-Asp, FLAG, a portion of an immunoglobulin protein,and a transcytosis peptide.

Fusion expression systems can be useful when it is desirable to producean immunogenic fragment of a polypeptide of the invention. For example,the VP6 capsid protein of rotavirus may be used as an immunologiccarrier protein for portions of polypeptide, either in the monomericform or in the form of a viral particle. The nucleic acid sequencescorresponding to the portion of a polypeptide of the invention to whichantibodies are to be raised may be incorporated into a fusion geneconstruct which includes coding sequences for a late vaccinia virusstructural protein to produce a set of recombinant viruses expressingfusion proteins comprising a portion of the protein as part of thevirion. The Hepatitis B surface antigen may also be utilized in thisrole as well. Similarly, chimeric constructs coding for fusion proteinscontaining a portion of a polypeptide of the invention and thepoliovirus capsid protein may be created to enhance immunogenicity (see,for example, EP Publication NO: 0259149; and Evans et al., (1989) Nature339:385; Huang et al., (1988) J. Virol. 62:3855; and Schlienger et al.,(1992) J. Virol. 66:2).

Fusion proteins may facilitate the expression and/or purification ofproteins. For example, a polypeptide of the invention may be generatedas a glutathione-S-transferase (GST) fusion protein. Such GST fusionproteins may be used to simplify purification of a polypeptide of theinvention, such as through the use of glutathione-derivatized matrices(see, for example, Current Protocols in Molecular Biology, eds. Ausubelet al., (N.Y.: John Wiley & Sons, 1991)). In another embodiment, afusion gene coding for a purification leader sequence, such as apoly-(His)/enterokinase cleavage site sequence at the N-terminus of thedesired portion of the recombinant protein, may allow purification ofthe expressed fusion protein by affinity chromatography using a Ni²⁺metal resin. The purification leader sequence may then be subsequentlyremoved by treatment with enterokinase to provide the purified protein(e.g., see Hochuli et al., (1987) J. Chromatography 411: 177; andJanknecht et al., PNAS USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene may be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments may be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which maysubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

In other embodiments, the invention provides for nucleic acids of theinvention immobilized onto a solid surface, including, plates,microtiter plates, slides, beads, particles, spheres, films, strands,precipitates, gels, sheets, tubing, containers, capillaries, pads,slices, etc. The nucleic acids of the invention may be immobilized ontoa chip as part of an array. The array may contain one or morepolynucleotides of the invention as described herein. In one embodiment,the chip contains one or more polynucleotides of the invention as partof an array of polynucleotide sequences from the same pathogenic speciesas such polynucleotide(s).

In a preferred form of the invention there is provided isolated B.hyodysenteriae polypeptides as herein described, and also thepolynucleotide sequences encoding these polypeptides. More desirably theB. hyodysenteriae polypeptides are provided in substantially purifiedform.

Preferred polypeptides of the invention will have one or more biologicalproperties (e.g., in vivo, in vitro or immunological properties) of thenative full-length polypeptide. Non-functional polypeptides are alsoincluded within the scope of the invention because they may be useful,for example, as antagonists of the functional polypeptides. Thebiological properties of analogues, fragments, or derivatives relativeto wild type may be determined, for example, by means of biologicalassays.

Polypeptides, including analogues, fragments and derivatives, can beprepared synthetically (e.g., using the well known techniques of solidphase or solution phase peptide synthesis). Preferably, solid phasesynthetic techniques are employed. Alternatively, the polypeptides ofthe invention can be prepared using well known genetic engineeringtechniques, as described infra. In yet another embodiment, thepolypeptides can be purified (e.g., by immunoaffinity purification) froma biological fluid, such as but not limited to plasma, faeces, serum, orurine from animals, including, but not limited to, pig, chicken, goose,duck, turkey, parakeet, human, monkey, dog, cat, horse, hamster, gerbil,rabbit, ferret, horse, cattle, and sheep.

The B. hyodysenteriae polypeptide analogues include those polypeptideshaving the amino acid sequence, wherein one or more of the amino acidsare substituted with another amino acid which substitutions do notsubstantially alter the biological activity of the molecule.

According to the invention, the polypeptides of the invention producedrecombinantly or by chemical synthesis and fragments or otherderivatives or analogues thereof, including fusion proteins, may be usedas an immunogen to generate antibodies that recognize the polypeptides.

A molecule is “antigenic” when it is capable of specifically interactingwith an antigen recognition molecule of the immune system, such as animmunoglobulin (antibody) or T cell antigen receptor. An antigenic aminoacid sequence contains at least about 5, and preferably at least about10, amino acids. An antigenic portion of a molecule can be the portionthat is immunodominant for antibody or T cell receptor recognition, orit can be a portion used to generate an antibody to the molecule byconjugating the antigenic portion to a carrier molecule forimmunization. A molecule that is antigenic need not be itselfimmunogenic, i.e., capable of eliciting an immune response without acarrier.

An “antibody” is any immunoglobulin, including antibodies and fragmentsthereof, that binds a specific epitope. The term encompasses polyclonal,monoclonal, and chimeric antibodies, the last mentioned described infurther detail in U.S. Pat. Nos. 4,816,397 and 4,816,567, as well asantigen binding portions of antibodies, including Fab, F(ab′)₂ and F(v)(including single chain antibodies). Accordingly, the phrase “antibodymolecule” in its various grammatical forms as used herein contemplatesboth an intact immunoglobulin molecule and an immunologically activeportion of an immunoglobulin molecule containing the antibody combiningsite. An “antibody combining site” is that structural portion of anantibody molecule comprised of heavy and light chain variable andhypervariable regions that specifically binds an antigen.

Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contain the paratope, including thoseportions known in the art as Fab, Fab′, F(ab′)₂ and F(v), which portionsare preferred for use in the therapeutic methods described herein.

Fab and F(ab′)₂ portions of antibody molecules are prepared by theproteolytic reaction of papain and pepsin, respectively, onsubstantially intact antibody molecules by methods that are well-known.See for example, U.S. Pat. No. 4,342,566 to Theofilopolous et al. Fab′antibody molecule portions are also well-known and are produced fromF(ab′)₂ portions followed by reduction with mercaptoethanol of thedisulfide bonds linking the two heavy chain portions, and followed byalkylation of the resulting protein mercaptan with a reagent such asiodoacetamide. An antibody containing intact antibody molecules ispreferred herein.

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may thereforecontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bispecific(chimeric) monoclonal antibody.

The term “adjuvant” refers to a compound or mixture that enhances theimmune response to an antigen. An adjuvant can serve as a tissue depotthat slowly releases the antigen and also as a lymphoid system activatorthat non-specifically enhances the immune response [Hood et al., inImmunology, p. 384, Second Ed., Benjamin/Cummings, Menlo Park, Calif.(1984)]. Often, a primary challenge with an antigen alone, in theabsence of an adjuvant, will fail to elicit a humoral or cellular immuneresponse. Adjuvants include, but are not limited to, complete Freund'sadjuvant, incomplete Freund's adjuvant, saponin, mineral gels such asaluminium hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Preferably, the adjuvant is pharmaceutically acceptable.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to the polypeptides of the invention. For theproduction of antibody, various host animals can be immunised byinjection with the polypeptide of the invention, including but notlimited to rabbits, mice, rats, sheep, goats, etc. In one embodiment, apolypeptide of the invention can be conjugated to an immunogeniccarrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin(KLH). Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole Limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward a polypeptideof the invention, any technique that provides for the production ofantibody molecules by continuous cell lines in culture may be used.These include but are not limited to the hybridoma technique originallydeveloped by Kohler et al., (1975) Nature, 256:495-497, the triomatechnique, the human B-cell hybridoma technique [Kozbor et al., (1983)Immunology Today, 4:72], and the EBV-hybridoma technique to producehuman monoclonal antibodies [Cole et al., (1985) in MonoclonalAntibodies and Cancer Therapy, pp. 77-96, Alan R. Liss, Inc.]. Immortal,antibody-producing cell lines can be created by techniques other thanfusion, such as direct transformation of B lymphocytes with oncogenicDNA, or transfection with Epstein-Barr virus. See, e.g., U.S. Pat. Nos.4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917;4,472,500; 4,491,632; and 4,493,890.

In an additional embodiment of the invention, monoclonal antibodies canbe produced in germ-free animals utilising recent technology. Accordingto the invention, chicken or swine antibodies may be used and can beobtained by using chicken or swine hybridomas or by transforming B cellswith EBV virus in vitro. In fact, according to the invention, techniquesdeveloped for the production of “chimeric antibodies” [Morrison et al.,(1984) J. Bacteriol., 159-870; Neuberger et al., (1984) Nature,312:604-608; Takeda et al., (1985) Nature, 314:452-454] by splicing thegenes from a mouse antibody molecule specific for a polypeptide of theinvention together with genes from an antibody molecule of appropriatebiological activity can be used; such antibodies are within the scope ofthis invention. Such chimeric antibodies are preferred for use intherapy of intestinal diseases or disorders (described infra), since theantibodies are much less likely than xenogenic antibodies to induce animmune response, in particular an allergic response, themselves.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce single chain antibodies specific for an polypeptide of theinvention. An additional embodiment of the invention utilises thetechniques described for the construction of Fab expression libraries[Huse et al., (1989) Science, 246:1275-1281] to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificityfor a polypeptide of the invention.

Antibody fragments, which contain the idiotype of the antibody molecule,can be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA, “sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitin reactions, immunodiffusion assays, in situ immunoassays(using colloidal gold, enzyme or radioisotope labels, for example),Western blots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays), complement fixationassays, immunofluorescence assays, protein A assays,immunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labelled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention. For example, to select antibodies thatrecognise a specific epitope of a polypeptide of the invention, one mayassay generated hybridomas for a product that binds to a fragment of apolypeptide of the invention containing such epitope.

The invention also covers diagnostic and prognostic methods to detectthe presence of B. hyodysenteriae using a polypeptide of the inventionand/or antibodies which bind to the polypeptide of the invention andkits useful for diagnosis and prognosis of B. hyodysenteriae infections.

Diagnostic and prognostic methods will generally be conducted using abiological sample obtained from an animal, such as chicken or swine. A“sample” refers to an animal's tissue or fluid suspected of containing aBrachyspira species, such as B. hyodysenteriae, or its polynucleotidesor its polypeptides. Examples of such tissue or fluids include, but notlimited to, plasma, serum, faecal material, urine, lung, heart, skeletalmuscle, stomach, intestines, and in vitro cell culture constituents.

The invention provides methods for detecting the presence of apolypeptide of the invention in a sample, with the following steps: (a)contacting a sample suspected of containing a polypeptide of theinvention with an antibody (preferably bound to a solid support) thatspecifically binds to the polypeptide of the invention under conditionswhich allow for the formation of reaction complexes comprising theantibody and the polypeptide of the invention; and (b) detecting theformation of reaction complexes comprising the antibody and polypeptideof the invention in the sample, wherein detection of the formation ofreaction complexes indicates the presence of the polypeptide of theinvention in the sample.

Preferably, the antibody used in this method is derived from anaffinity-purified polyclonal antibody, and more preferably a monoclonalantibody. In addition, it is preferable for the antibody molecules usedherein be in the form of Fab, Fab′, F(ab′)₂ or F(v) portions or wholeantibody molecules.

Particularly preferred methods for detecting B. hyodysenteriae based onthe above method include enzyme linked immunosorbent assays,radioimmunoassays, immunoradiometric assays and immunoenzymatic assays,including sandwich assays using monoclonal and/or polyclonal antibodies.

Three such procedures that are especially useful utilise eitherpolypeptide of the invention (or a fragment thereof) labelled with adetectable label, antibody Ab₁ labelled with a detectable label, orantibody Ab₂ labelled with a detectable label. The procedures may besummarized by the following equations wherein the asterisk indicatesthat the particle is labelled and “AA” stands for the polypeptide of theinvention:AA*+Ab ₁ =AA*Ab ₁  A.AA+Ab* ₁ =AAAb ₁*  B.AA+Ab ₁ +Ab ₂ *=Ab ₁ AAAb ₂*  C.

The procedures and their application are all familiar to those skilledin the art and accordingly may be utilised within the scope of thepresent invention. The “competitive” procedure, Procedure A, isdescribed in U.S. Pat. Nos. 3,654,090 and 3,850,752. Procedure B isrepresentative of well-known competitive assay techniques. Procedure C,the “sandwich” procedure, is described in U.S. Pat. Nos. RE 31,006 and4,016,043. Still other procedures are known, such as the “doubleantibody” or “DASP” procedure, and can be used.

In each instance, the polypeptide of the invention form complexes withone or more antibody(ies) or binding partners and one member of thecomplex is labelled with a detectable label. The fact that a complex hasformed and, if desired, the amount thereof, can be determined by knownmethods applicable to the detection of labels.

It will be seen from the above, that a characteristic property of Ab₂ isthat it will react with Ab₁. This reaction is because Ab₁, raised in onemammalian species, has been used in another species as an antigen toraise the antibody, Ab₂. For example, Ab₂ may be raised in goats usingrabbit antibodies as antigens. Ab₂ therefore would be anti-rabbitantibody raised in goats. For purposes of this description and claims,Ab₁ will be referred to as a primary antibody, and Ab₂ will be referredto as a secondary or anti-Ab₁ antibody.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals that fluoresce when exposed to ultravioletlight, and others. Examples of fluorescent materials capable of beingutilised as labels include fluorescein, rhodamine and auramine. Aparticular detecting material is anti-rabbit antibody prepared in goatsand conjugated with fluorescein through an isothiocyanate. Examples ofpreferred isotope include ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co,⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re. The radioactive label can be detectedby any of the currently available counting procedures. While manyenzymes can be used, examples of preferred enzymes are peroxidase,β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucoseoxidase plus peroxidase and alkaline phosphatase. Enzyme are conjugatedto the selected particle by reaction with bridging molecules such ascarbodiimides, diisocyanates, glutaraldehyde and the like. Enzyme labelscan be detected by any of the presently utilized colorimetric,spectrophotometric, fluorospectrophotometric, amperometric or gasometrictechniques. U.S. Pat. Nos. 3,654,090; 3,850,752; and 4,016,043 arereferred to by way of example for their disclosure of alternatelabelling material and methods.

The invention also provides a method of detecting antibodies to apolypeptide of the invention in biological samples, using the followingsteps: (a) providing a polypeptide of the invention or a fragmentthereof; (b) incubating a biological sample with said polypeptide of theinvention under conditions which allow for the formation of anantibody-antigen complex; and (c) determining whether anantibody-antigen complex with the polypeptide of the invention isformed.

In another embodiment of the invention there are provided in vitromethods for evaluating the level of antibodies to a polypeptide of theinvention in a biological sample using the following steps: (a)detecting the formation of reaction complexes in a biological sampleaccording to the method noted above; and (b) evaluating the amount ofreaction complexes formed, which amount of reaction complexescorresponds to the level of polypeptide of the invention in thebiological sample.

Further there are provided in vitro methods for monitoring therapeutictreatment of a disease associated with B. hyodysenteriae in an animalhost by evaluating, as describe above, the levels of antibodies to apolypeptide of the invention in a series of biological samples obtainedat different time points from an animal host undergoing such therapeutictreatment.

The present invention further provides methods for detecting thepresence or absence of B. hyodysenteriae in a biological sample by: (a)bringing the biological sample into contact with a polynucleotide probeor primer of polynucleotide of the invention under suitable hybridizingconditions; and (b) detecting any duplex formed between the probe orprimer and nucleic acid in the sample.

According to one embodiment of the invention, detection of B.hyodysenteriae may be accomplished by directly amplifying polynucleotidesequences from biological sample, using known techniques and thendetecting the presence of polynucleotide of the invention sequences.

In one form of the invention, the target nucleic acid sequence isamplified by PCR and then detected using any of the specific methodsmentioned above. Other useful diagnostic techniques for detecting thepresence of polynucleotide sequences include, but are not limited to: 1)allele-specific PCR; 2) single stranded conformation analysis; 3)denaturing gradient gel electrophoresis; 4) RNase protection assays; 5)the use of proteins which recognize nucleotide mismatches, such as theE. coli mutS protein; 6) allele-specific oligonucleotides; and 7)fluorescent in situ hybridisation.

In addition to the above methods polynucleotide sequences may bedetected using conventional probe technology. When probes are used todetect the presence of the desired polynucleotide sequences, thebiological sample to be analysed, such as blood or serum, may betreated, if desired, to extract the nucleic acids. The samplepolynucleotide sequences may be prepared in various ways to facilitatedetection of the target sequence; e.g. denaturation, restrictiondigestion, electrophoresis or dot blotting. The targeted region of thesample polynucleotide sequence usually must be at least partiallysingle-stranded to form hybrids with the targeting sequence of theprobe. If the sequence is naturally single-stranded, denaturation willnot be required. However, if the sequence is double-stranded, thesequence will probably need to be denatured. Denaturation can be carriedout by various techniques known in the art.

Sample polynucleotide sequences and probes are incubated underconditions that promote stable hybrid formation of the target sequencein the probe with the putative desired polynucleotide sequence in thesample. Preferably, high stringency conditions are used in order toprevent false positives.

Detection, if any, of the resulting hybrid is usually accomplished bythe use of labelled probes. Alternatively, the probe may be unlabeled,but may be detectable by specific binding with a ligand that islabelled, either directly or indirectly. Suitable labels and methods forlabelling probes and ligands are known in the art, and include, forexample, radioactive labels which may be incorporated by known methods(e.g., nick translation, random priming or kinasing), biotin,fluorescent groups, chemiluminescent groups (e.g., dioxetanes,particularly triggered dioxetanes), enzymes, antibodies and the like.Variations of this basic scheme are known in the art, and include thosevariations that facilitate separation of the hybrids to be detected fromextraneous materials and/or that amplify the signal from the labelledmoiety.

It is also contemplated within the scope of this invention that thenucleic acid probe assays of this invention may employ a cocktail ofnucleic acid probes capable of detecting the desired polynucleotidesequences of this invention. Thus, in one example to detect the presenceof polynucleotide sequences of this invention in a cell sample, morethan one probe complementary to a polynucleotide sequences is employedand in particular the number of different probes is alternatively 2, 3,or 5 different nucleic acid probe sequences.

The polynucleotide sequences described herein (preferably in the form ofprobes) may also be immobilised to a solid phase support for thedetection of Brachyspira species, including but not limited to B.hyodysenteriae, B. intermedia, B. alvinipulli, B. aalborgi, B. innocens,B. murdochii, and B. pilosicoli. Alternatively the polynucleotidesequences described herein will form part of a library of DNA moleculesthat may be used to detect simultaneously a number of different genesfrom Brachyspira species, such as B. hyodysenteriae. In a furtheralternate form of the invention polynucleotide sequences describedherein together with other polynucleotide sequences (such as from otherbacteria or viruses) may be immobilised on a solid support in such amanner permitting identification of the presence of a Brachyspiraspecies, such as B. hyodysenteriae and/or any of the otherpolynucleotide sequences bound onto the solid support.

Techniques for producing immobilised libraries of DNA molecules havebeen described in the art. Generally, most prior art methods describethe synthesis of single-stranded nucleic acid molecule libraries, usingfor example masking techniques to build up various permutations ofsequences at the various discrete positions on the solid substrate. U.S.Pat. No. 5,837,832 describes an improved method for producing DNA arraysimmobilised to silicon substrates based on very large scale integrationtechnology. In particular, U.S. Pat. No. 5,837,832 describes a strategycalled “tiling” to synthesize specific sets of probes at spatiallydefined locations on a substrate that may be used to produced theimmobilised DNA libraries of the present invention. U.S. Pat. No.5,837,832 also provides references for earlier techniques that may alsobe used. Thus polynucleotide sequence probes may be synthesised in situon the surface of the substrate.

Alternatively, single-stranded molecules may be synthesised off thesolid substrate and each pre-formed sequence applied to a discreteposition on the solid substrate. For example, polynucleotide sequencesmay be printed directly onto the substrate using robotic devicesequipped with either pins or pizo electric devices.

The library sequences are typically immobilised onto or in discreteregions of a solid substrate. The substrate may be porous to allowimmobilisation within the substrate or substantially non-porous, inwhich case the library sequences are typically immobilised on thesurface of the substrate. The solid substrate may be made of anymaterial to which polypeptides can bind, either directly or indirectly.Examples of suitable solid substrates include flat glass, siliconwafers, mica, ceramics and organic polymers such as plastics, includingpolystyrene and polymethacrylate. It may also be possible to usesemi-permeable membranes such as nitrocellulose or nylon membranes,which are widely available. The semi-permeable membranes may be mountedon a more robust solid surface such as glass. The surfaces mayoptionally be coated with a layer of metal, such as gold, platinum orother transition metal.

Preferably, the solid substrate is generally a material having a rigidor semi-rigid surface. In preferred embodiments, at least one surface ofthe substrate will be substantially flat, although in some embodimentsit may be desirable to physically separate synthesis regions fordifferent polymers with, for example, raised regions or etched trenches.It is also preferred that the solid substrate is suitable for the highdensity application of DNA sequences in discrete areas of typically from50 to 100 μm, giving a density of 10000 to 40000 dots/cm⁻².

The solid substrate is conveniently divided up into sections. This maybe achieved by techniques such as photoetching, or by the application ofhydrophobic inks, for example teflon-based inks (Cel-line, USA).

Discrete positions, in which each different member of the library islocated may have any convenient shape, e.g., circular, rectangular,elliptical, wedge-shaped, etc.

Attachment of the polynucleotide sequences to the substrate may be bycovalent or non-covalent means. The polynucleotide sequences may beattached to the substrate via a layer of molecules to which the librarysequences bind. For example, the polynucleotide sequences may belabelled with biotin and the substrate coated with avidin and/orstreptavidin. A convenient feature of using biotinylated polynucleotidesequences is that the efficiency of coupling to the solid substrate canbe determined easily. Since the polynucleotide sequences may bind onlypoorly to some solid substrates, it is often necessary to provide achemical interface between the solid substrate (such as in the case ofglass) and the nucleic acid sequences. Examples of suitable chemicalinterfaces include hexaethylene glycol. Another example is the use ofpolylysine coated glass, the polylysine then being chemically modifiedusing standard procedures to introduce an affinity ligand. Other methodsfor attaching molecules to the surfaces of solid substrate by the use ofcoupling agents are known in the art, see for example WO98/49557.

Binding of complementary polynucleotide sequences to the immobilisednucleic acid library may be determined by a variety of means such aschanges in the optical characteristics of the bound polynucleotidesequence (i.e. by the use of ethidium bromide) or by the use of labellednucleic acids, such as polypeptides labelled with fluorophores. Otherdetection techniques that do not require the use of labels includeoptical techniques such as optoacoustics, reflectometry, ellipsometryand surface plasmon resonance (see WO97/49989).

Thus, the present invention provides a solid substrate havingimmobilized thereon at least one polynucleotide of the presentinvention, preferably two or more different polynucleotide sequences ofthe present invention.

The present invention also can be used as a prophylactic or therapeutic,which may be utilised for the purpose of stimulating humoral and cellmediated responses in animals, such as chickens and swine, therebyproviding protection against colonisation with Brachyspira species,including but not limited to B. hyodysenteriae, B. intermedia, B.alvinipulli, B. aalborgi, B. innocens, B. murdochii, and B. pilosicoli.Natural infection with a Brachyspira species, such as B. hyodysenteriaeinduces circulating antibody titres against the proteins describedherein. Therefore, the amino acid sequences described herein or partsthereof, have the potential to form the basis of a systemically ororally administered prophylactic or therapeutic to provide protectionagainst intestinal spirochaetosis.

It is well appreciated by those skilled in the art there is a highdegree of sequence conservation between species of Brachyspira. Forexample, as shown below B. hyodysenteriae outer membrane proteins (OMP)H122 and H114 have a high degree of similarity between a number ofBrachyspira species.

Cross-species nucleotide similarity for B. hyodysenteriae OMP H114.

Similarity at nucleotide level (%) Brachyspira species H114 Brachyspirahyodysenteriae 100 Brachyspira pilosicoli 85.4 Brachyspira intermedia93.9 Brachyspira murdochii 90.8 Brachyspira alvinpulli 93.8

Cross-species nucleotide similarity for B. hyodysenteriae OMP H122.

Similarity at nucleotide level (%) Brachyspira species H122 Brachyspirahyodysenteriae 100 Brachyspira pilosicoli 81.4 Brachyspira intermedia87.5 Brachyspira murdochii 83.0 Brachyspira alvinipulli 82.9

Also as shown in Example 22, the nucleotide and amino acid sequences ofthe present invention are capable of producing cross-reactivity over arange of Brachyspira species not just B. hyodysenteriae. Accordingly,polypeptides used in the present invention are capable of use againstother species of Brachyspira species not just B. hyodysenteriae.

Accordingly, in one embodiment the present invention provides the aminoacid sequences described herein or fragments thereof or antibodies thatbind the amino acid sequences or the polynucleotide sequences describedherein in a therapeutically effective amount admixed with apharmaceutically acceptable carrier, diluent, or excipient.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to reduce by at least about 15%, preferably by atleast 50%, more preferably by at least 90%, and most preferably prevent,a clinically significant deficit in the activity, function and responseof the animal host. Alternatively, a therapeutically effective amount issufficient to cause an improvement in a clinically significant conditionin the animal host.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similarly untoward reaction, such as gastricupset and the like, when administered to an animal. The term “carrier”refers to a diluent, adjuvant, excipient, or vehicle with which thecompound is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water or saline solutions and aqueousdextrose and glycerol solutions are preferably employed as carriers,particularly for injectable solutions. Suitable pharmaceutical carriersare described in Martin, Remington's Pharmaceutical Sciences, 18th Ed.,Mack Publishing Co., Easton, Pa., (1990).

In a more specific form of the invention there are providedpharmaceutical compositions comprising therapeutically effective amountsof the amino acid sequences described herein or an analogue, fragment orderivative product thereof or antibodies thereto together withpharmaceutically acceptable diluents, preservatives, solubilizes,emulsifiers, adjuvants and/or carriers. Such compositions includediluents of various buffer content (e.g., Tris-HCl, acetate, phosphate),pH and ionic strength and additives such as detergents and solubilizingagents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbicacid, sodium metabisulfite), preservatives (e.g., Thimersol, benzylalcohol) and bulking substances (e.g., lactose, mannitol). The materialmay be incorporated into particulate preparations of polymeric compoundssuch as polylactic acid, polyglycolic acid, etc. or into liposomes.Hylauronic acid may also be used. Such compositions may influence thephysical state, stability, rate of in vivo release, and rate of in vivoclearance of the present proteins and derivatives. See, e.g., Martin,Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack PublishingCo., Easton, Pa. 18042) pages 1435-1712 that are herein incorporated byreference. The compositions may be prepared in liquid form, or may be indried powder, such as lyophilised form.

Alternatively, the polynucleotides of the invention can be optimized forexpression in plants (e.g., corn). The plant may be transformed withplasmids containing the optimized polynucleotides. Then the plant isgrown, and the proteins of the invention are expressed in the plant, orthe plant-optimized version is expressed. The plant is later harvested,and the section of the plant containing the proteins of the invention isprocessed into feed for the animal. This animal feed will impartimmunity against B. hyodysenteriae when eaten by the animal. Examples ofprior art detailing these methods can be found in U.S. Pat. No.5,914,123 (Arntzen, et al.); U.S. Pat. No. 6,034,298 (Lam, et al.); andU.S. Pat. No. 6,136,320 (Arntzen, et al.).

It will be appreciated that pharmaceutical compositions providedaccordingly to the invention may be administered by any means known inthe art. Preferably, the pharmaceutical compositions for administrationare administered by injection, orally, or by the pulmonary, or nasalroute. The amino acid sequences described herein or antibodies derivedtherefrom are more preferably delivered by intravenous, intraarterial,intraperitoneal, intramuscular, or subcutaneous routes ofadministration. Alternatively, the amino acid sequence described hereinor antibodies derived therefrom, properly formulated, can beadministered by nasal or oral administration.

In a specific embodiment of the invention, a vaccine composition may bea “combination vaccine” with one or more polynucleotide or polypeptidesequences of the invention which have a similar functional role that arecombined in a vaccine. For example, the following three differentfunctional groups can be combined as identified in the following tables:

Vaccine: 1

Polynucleotide sequence Polypeptide sequence Gene SEQ ID No. SEQ ID No.NAV-H77 SEQ ID No. 1 SEQ ID No. 2 NAV-H105 SEQ ID No. 3 SEQ ID No. 4NAV-H109 SEQ ID No. 5 SEQ ID No. 6 NAV-H114 SEQ ID No. 7 SEQ ID No. 8Vaccine: 2

Polynucleotide sequence Polypeptide sequence Gene SEQ ID No. SEQ ID No.NAV-H116 SEQ ID No. 9 SEQ ID No. 10 NAV-H122 SEQ ID No. 11 SEQ ID No. 12NAV-H147 SEQ ID No. 13 SEQ ID No. 14 NAV-H155 SEQ ID No. 15 SEQ ID No.16Vaccine: 3

Polynucleotide sequence Polypeptide sequence Gene SEQ ID No. SEQ ID No.NAV-H161 SEQ ID No. 17 SEQ ID No. 18 NAV-H173 SEQ ID No. 19 SEQ ID No.20

A prerequisite for combination vaccines is a lack of competition (i.e.between antigens) and a high compatibility with respect to the subjectto be immunized. According to international standard applied inconnection with immunization, a combination vaccine should confer aprotection which is comparable to that achieved by separatevaccinations. However, the combination of antigens is a complicatedprocess which is associated with a number of problems and uncertainties.

As the individual components of a combination vaccine are not inertsubstances, the combination of various components into one mixture cannegatively influence the immunogenicity of the individual antigeniccomponents. For example, interactions between the different antigens orbetween the antigens and other components typically used in suchvaccines can occur due to differences in charge, chemical residues,detergents, formaldehyde, concentration of ions etc. These changes canoccur instantaneously after contacting the different antigens with eachother or with other substances usually present in vaccine formulations.Moreover, these changes can also occur with a significant delay aftermixing, for example during storage, shipping, etc. However, it cannot bepredicted to which extent the immunogenicity of individual antigens maybe influenced by mixing them to one combination. As a consequence, asituation can occur in which one or more antigens of the vaccine onlyconfer an insufficient seroprotection against one or more of diseaseswhich renders the product unsuitable for medical practice.

The production of effective combination vaccines is a complex matterwhich depends on multidimensional interactions between the individualcomponents of the vaccine and does not allow to extrapolate any effectof the components observed when administered separately.

It has been found that the combination vaccines according to the presentinvention may be combined with one or more further antigens and remaineffective and stable.

The term “stable” as used in the context of the present invention meansthat the combination vaccine formulation can be kept for a period ofeight days or more preferably 14 days at room temperature without anysubstantial loss with respect to immunogenity and stability of itsdistinct antigen compounds. In order to enhance stability, stabilizingagents such as saccharose can be added to the vaccine composition.Instead of saccharose or in addition thereto, other stabilizers, e.g.human serum albumin, mannose, trehalose, mannite, or polygeline can alsobe added as stabilizing agents. The vaccines of the present inventionexhibit a pH value of preferably between 5.0 to 8.0, more preferablybetween 6.0 to 7.0, wherein a pH value of 6.8 to 7.8 is most preferred.

The term “effective” as used herein, refers to the fact that thesequence of the invention upon single or repeated administration to asubject (for example, a mammal) confers protection against a specificdisease.

As used herein the term “conferring protection” means that the sequenceof the invention, upon administration, induces an immunological responsein the vaccinated subject which response is capable to protect saidsubject from the symptoms of subsequent infection.

Also encompassed by the present invention is the use of polynucleotidesequences of the invention, as well as antisense and ribozymepolynucleotide sequences hybridisable to a polynucleotide sequenceencoding an amino acid sequence according to the invention, formanufacture of a medicament for modulation of a disease associated B.hyodysenteriae.

Polynucleotide sequences encoding antisense constructs or ribozymes foruse in therapeutic methods are desirably administered directly as anaked nucleic acid construct. Uptake of naked nucleic acid constructs bybacterial cells is enhanced by several known transfection techniques,for example those including the use of transfection agents. Example ofthese agents include cationic agents (for example calcium phosphate andDEAE-dextran) and lipofectants. Typically, nucleic acid constructs aremixed with the transfection agent to produce a composition.

Alternatively the antisense construct or ribozymes may be combined witha pharmaceutically acceptable carrier or diluent to produce apharmaceutical composition. Suitable carriers and diluents includeisotonic saline solutions, for example phosphate-buffered saline. Thecomposition may be formulated for parenteral, intramuscular,intravenous, subcutaneous, intraocular, oral or transdermaladministration. The routes of administration described are intended onlyas a guide since a skilled practitioner will be able to determinereadily the optimum route of administration and any dosage for anyparticular animal and condition.

The invention also includes kits for screening animals suspected ofbeing infected with a Brachyspira species, such as B. hyodysenteriae orto confirm that an animal is infected with a Brachyspira species, suchas B. hyodysenteriae. In a further embodiment of this invention, kitssuitable for use by a specialist may be prepared to determine thepresence or absence of Brachyspira species, including but not limited toB. hyodysenteriae, B. intermedia, B. alvinipulli, B. aalborgi, B.innocens, B. murdochii, and B. pilosicoli in suspected infected animalsor to quantitatively measure a Brachyspira species, including but notlimited to B. hyodysenteriae, B. intermedia, B. alvinipulli, B. aalborgiand B. pilosicoli infection. In accordance with the testing techniquesdiscussed above, such kits can contain at least a labelled version ofone of the amino acid sequences described herein or its binding partner,for instance an antibody specific thereto, and directions depending uponthe method selected, e.g., “competitive,” “sandwich,” “DASP” and thelike. Alternatively, such kits can contain at least a polynucleotidesequence complementary to a portion of one of the polynucleotidesequences described herein together with instructions for its use. Thekits may also contain peripheral reagents such as buffers, stabilizers,etc.

Accordingly, a test kit for the demonstration of the presence of aBrachyspira species, including but not limited to B. hyodysenteriae, B.intermedia, B. alvinipulli, B. aalborgi, B. innocens, B. murdochii, andB. pilosicoli, may contain the following:

(a) a predetermined amount of at least one labelled immunochemicallyreactive component obtained by the direct or indirect attachment of oneof the amino acid sequences described herein or a specific bindingpartner thereto, to a detectable label;

(b) other reagents; and

(c) directions for use of said kit.

More specifically, the diagnostic test kit may contain:

(a) a known amount of one of the amino acid sequences described hereinas described above (or a binding partner) generally bound to a solidphase to form an immunosorbent, or in the alternative, bound to asuitable tag, or there are a plural of such end products, etc;

(b) if necessary, other reagents; and

(c) directions for use of said test kit.

In a further variation, the test kit may contain:

(a) a labelled component which has been obtained by coupling one of theamino acid sequences described herein to a detectable label;

(b) one or more additional immunochemical reagents of which at least onereagent is a ligand or an immobilized ligand, which ligand is selectedfrom the group consisting of:

-   -   (i) a ligand capable of binding with the labelled component (a);    -   (ii) a ligand capable of binding with a binding partner of the        labelled component (a);    -   (iii) a ligand capable of binding with at least one of the        component(s) to be determined; or    -   (iv) a ligand capable of binding with at least one of the        binding partners of at least one of the component(s) to be        determined; and

(c) directions for the performance of a protocol for the detectionand/or determination, of one or more components of an immunochemicalreaction between one of the amino acid sequences described herein and aspecific binding partner thereto.

In one embodiment, the present invention provides simple point-of-carekit that uses principles similar to the widely-used serum testing kits,for the rapid detection of the circulating antibodies in the animal tothe polypeptides of the invention will allow the healthcare professionalor veterinarian to rapidly diagnose Brachyspira infection, and torapidly institute proven and effective therapeutic and preventivemeasures. The use of the kit can represent the standard of care for allanimals that are at risk of developing Brachyspira infection.

The methods and kits of the present invention can also provide a meansfor detecting or monitoring Brachyspira infection including the changein status. It can be especially useful in detecting early stageBrachyspira infection. Thus, the invention also provides a means for ahealthcare professional or veterinarian to monitor the progression ofBrachyspira infection (worsening, improving, or remaining the same)during and following treatment. Typically, the healthcare professionalor veterinarian would establish a protocol of collecting and analysing aquantity of biological sample from the animal at selected intervals.Typically the sample is obtained intermittently during a prescribedperiod. The period of time between intermittent sampling may be dictatedby the condition of the animal, and can range from a sample each 24hours to a sample taken days, weeks or even months apart.

A point-of-care kit for use in the method typically comprises a mediahaving affixed thereto one or more capture polypeptides, whereby thesample is contacted with the media to expose the capture polypeptides tothe circulating antibodies contained in the sample. The kit includes anacquiring means that can comprise an implement, such as a needle orvacutainer, having a surface comprising the media. The acquiring meanscan also comprise a container for accepting the sample, where thecontainer has a serum-contacting surface that comprises the media. Inanother typical embodiment, the assay for detecting the complex of thecirculating antibodies and the capture polypeptides can comprise anELISA, and can be used to quantitate the amount of circulatingantibodies to the polypeptides of the invention in a sample. In analternative embodiment, the acquiring means can comprise an implementcomprising a cassette containing the media.

A method and kit of the present invention for detecting the circulatingantibodies to the polypeptides of the invention can be made by adaptingthe methods and kits known in the art for the rapid detection of otherproteins and ligands in a biological sample. Examples of methods andkits that can be adapted to the present invention are described in U.S.Pat. No. 5,656,503, issued to May et al. on Aug. 12, 1997, U.S. Pat. No.6,500,627, issued to O'Conner et al. on Dec. 31, 2002, U.S. Pat. No.4,870,007, issued to Smith-Lewis on Sep. 26, 1989, U.S. Pat. No.5,273,743, issued to Ahlem et al. on Dec. 28, 1993, and U.S. Pat. No.4,632,901, issued to Valkers et al. on Dec. 30, 1986, all suchreferences being hereby incorporated by reference.

A rapid one-step method of detecting the circulating antibodies to thepolypeptides of the present invention can reduce the time for detectingthe development of Brachyspira infection. A typical method can comprisethe steps of: obtaining a sample from an animal at risk of or suspectedof having a Brachyspira infection; mixing a portion of the sample withone or more detecting polypeptides which specifically bind to one of thecirculating antibodies, so as to initiate the binding of the detectingpolypeptides to the circulating antibodies in the sample; contacting themixture of sample and detecting polypeptides with an immobilized captureantibody which specifically binds to the detecting polypeptides, whichcapture antibody does not cross-react with the detecting polypeptide, soas to bind the detecting polypeptide to the circulating antibodies, andthe circulating antibodies to the capture antibody, to form a detectablecomplex; removing unbound detecting polypeptide and any unbound samplefrom the complex; and detecting the detecting polypeptide of thecomplex. The detectable polypeptide can be labelled with a detectablemarker, such as a radioactive label, enzyme, biological dye, magneticbead, or biotin, as is well known in the art.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the invention.

Example 1 Genome Sequencing

An Australian porcine field isolate of B. hyodysenteriae (strain WA1) isshotgun sequenced. This strain has been well-characterised and shown tobe virulent following experimental challenge of pigs. The spirochaete isgrown in anaerobic trypticase soy broth culture and 100 μg DNA wasextracted using a cetyltrimethylammonium bromide (CTAB) method toprepare high quality chromosomal DNA suitable for preparation of genomicDNA libraries. The genomic DNA is sheared using a GeneMachinesHydroshear, and the fragmented DNA processed for cloning as per theprotocol recommended by the suppliers of the pSMART vector system(Lucigen). A small insert (2-3 kb) library and a medium insert (3-10 kb)library are constructed into the low copy version of the pSMART vector.

Example 2 Annotation

Partial genome sequences for B. hyodysenteriae are assembled andannotated by the Australian Genome Research Facility (AGRF) inQueensland and at Murdoch University by the Centre for Bioinformaticsand Biological Computing (CBBC). The CBBC uses up-to-date mirrors of themajor international databases and have developed leading edgebioinformatics software and strategies for annotation of features inlarge genome sequences. A range of public domain bioinformatics toolsare used to analyse and re-analyse the sequences as part of a qualityassurance procedure on data analysis. Open reading frames (ORFs) arepredicted using a variety of programs including GeneMark, GLIMMER,ORPHEUS, SELFID and GetORF. Putative ORFs are examined for homology (DNAand protein) with existing international databases using searchesincluding BLAST and FASTA. All the predicted ORFs are analysed todetermine their cellular localisation using programs such as PSI-BLAST,FASTA, MOTIFS, FINDPATTERNS, PHD, SIGNALP and PSORT. Databases includingInterpro, Prosite, Propom, Pfam and Blocks are used to predict surfaceassociated proteins such as transmembrane domains, leader peptides,homologies to know surface proteins, lipoprotein signature, outermembrane anchoring motifs and host cell binding domains. Phylogeneticand other molecular evolution analysis is conducted with the identifiedgenes and with other species to assist in the assignment of function.The in silico analysis of both partially sequenced genomes has produceda comprehensive list of all the predicted ORFs present in the sequencedata available. Each ORF is interrogated for descriptive informationsuch as predicted molecular weight, isoelectric point, hydrophobicity,and subcellular localisation to enable correlation with the in vitroproperties of the native gene product. Predicted genes which encodeproteins similar to surface localized components and virulence factorsin other pathogenic bacteria are selected as potential vaccine targets.

Example 3 Analysis of Gene Distribution Using PCR

One or two primer pairs which anneal to different regions of the targetgene coding region are designed and optimised for PCR detection.Distribution analysis of the B. hyodysenteriae target genes is performedon 23 strains of B. hyodysenteriae, including two strains which havebeen shown to be avirulent. Primer sets used in the distributionanalysis are shown in Table 1. PCR analysis is performed in a 25 μltotal volume using Taq DNA polymerase. The amplification mixtureconsisted of 1×PCR buffer (containing 1.5 mM of MgCl2), 1 U of Taq DNApolymerase, 0.2 mM of each dNTP, 0.5 μM of the primer pair, and 1 μlpurified chromosomal template DNA. Cycling conditions involved aninitial template denaturation step of 5 min at 94° C., followed by 30cycles of denaturation at 94° C. for 30 s, annealing at 50° C. for 15 s,and primer extension at 72° C. for 1 min. The PCR products are subjectedto electrophoresis in 1% (w/v) agarose gels in 1×TAE buffer (40 mMTris-acetate, 1 mM EDTA), stained with a 1 μg/ml ethidium bromidesolution and viewed over ultraviolet (UV) light.

TABLE 1 OLIGONUCLEOTIDE PRIMERS USED IN THE DISTRIBUTION ANALYSISOF THE B. HYODYSENTERIAE VACCINE CANDIDATE GENES Primer Gene namePrimer Sequence (5′-3′) SEQ ID No. NAV-H77 H77-F26GCTTATTTACTATGGTGTCGGCATTAG SEQ ID No. 21 H77-R696ATATCTTTCTTCTTCTTCGTCTTCTTC SEQ ID No. 22 NAV-H105 H105-F286AGAATACCTCTTTCACGCGGACTTGGA SEQ ID No. 23 H105-R456 ACCTCCCAATATTGCAGGAGSEQ ID No. 24 NAV-H109 H109-F64 TTTGTTATGGGCTTTGTAGG SEQ ID No. 25H109-R513 AAGAAGAAAATCTGCTGAAAC SEQ ID No. 26 NAV-H114 H114-F46ATTCAATGCGGTAATAAAACAGATAC SEQ ID No. 27 H114-R650ATGCTAATATCCCCTACTTCTTCAAG SEQ ID No. 28 NAV-H116 H116-F210TGAATCGGCTGTAAAGCAGGCA SEQ ID No. 29 H116-R266ACACCCGCATCAAATCCAGCACCTGA SEQ ID No. 30 NAV-H122 H122-F54TCTTATGGCTAAAAGCGGATTCGGA SEQ ID No. 31 H122-R385GAGGGAATTTTACACCTGCTCCAACACC SEQ ID No. 32 NAV-H147 H147-F185TCGCTGAAGGTTATGCATCTGC SEQ ID No. 33 H147-R489TCTGTCCATCAGTATGCCCATTGCCTGA SEQ ID No. 34 NAV-H155 H155-F64CAATTTGATGCTAGCATATATGCAC SEQ ID No. 35 H155-R760AATTTAAAGCAATTTCTAAACTGCTAAATC SEQ ID No. 36 NAV-H161 H161-F124AGCAGATCATACTCTATAAAAACAGG SEQ ID No. 37 H161-R586GTCTATTAGCAAATAAGAACTCCAATG SEQ ID No. 38 NAV-H173 H173-F272TAAAAGGAGAAAAAGGAAGATACG SEQ ID No. 39 H179-R705 ATTATCTTGATGAGGATGCTTTCSEQ ID No. 40

Example 4 Bioinformatics

Shot-gun sequencing of the B. hyodysenteriae genome resulted in 73%(2,347.8 kb out of a predicted 2,300 kb) of the genome to be sequenced.These sequences are comprised of 171 contigs with an average contig sizeof 13.7 kb. From the 171 contigs, 1,860 open-reading frames (ORFs) arepredicted. Comparison of the predicted ORFs with genes present in thenucleic acid and protein databases indicated that approximately 70% ineach species have homology with genes contained in the databases. Theremaining 30% of the predicted ORFs from each genome have no knownidentity.

Example 5 Vaccine Candidates

The gene products of the predicted ORFs were analysed using the PSORTbSubcellular Localisation Prediction Tool (Gardy, J. L., Laird, M. R.,Chen, F., Rey, S., Walsh, C. J., Ester, M. and Brinkman, F. S. L. (2005)PSORTb v.2.0: expanded prediction of bacterial protein subcellularlocalization and insights gained from comparative proteome analysis.Bioinformatics 21:617-623.] to determine the probable subcellularlocalisation of the protein. Proteins with a significant probability ofhaving an extracellular or outer membrane localisation were selected aspotential candidate antigens for vaccine and serology. Table 2 providesthe basic descriptive information associated with each selected gene andits putative gene product.

TABLE 2 DESCRIPTIVE INFORMATION ASSOCIATED WITH THE B. HYODYSENTERIAEGENES AND PUTATIVE GENE PRODUCTS PREDICTED BY PSORTB TO HAVE AN OUTERMEMBRANE LOCALISATION Gene Protein size size Predicted Predicted GeneSEQ ID Nos. (bp) (aa) MW (Da) pI NAV-H77 Nucleotide seq. SEQ ID No. 1702 234 27,291 4.8096 Amino acid seq. SEQ ID No. 2 NAV-H105 Nucleotideseq. SEQ ID No. 3 942 314 35,447 9.9264 Amino acid seq. SEQ ID No. 4NAV-H109 Nucleotide seq. SEQ ID No. 5 675 225 26,275 5.3190 Amino acidseq. SEQ ID No. 6 NAV-H114 Nucleotide seq. SEQ ID No. 7 996 332 39,1994.0603 Amino acid seq. SEQ ID No. 8 NAV-H116 Nucleotide seq. SEQ ID No.9 810 270 29,654 9.9131 Amino acid seq. SEQ ID No. 10 NAV-H122Nucleotide seq. SEQ ID No. 11 639 213 24,082 5.2678 Amino acid seq. SEQID No. 12 NAV-H147 Nucleotide seq. SEQ ID No. 13 1239 413 45,175 7.4703Amino acid seq. SEQ ID No. 14 NAV-H155 Nucleotide seq. SEQ ID No. 15 759253 28,879 5.2134 Amino acid seq. SEQ ID No. 16 NAV-H161 Nucleotide seq.SEQ ID No. 17 792 264 32,298 4.6577 Amino acid seq. SEQ ID No. 18NAV-H173 Nucleotide seq. SEQ ID No. 19 639 213 23,789 8.3429 Amino acidseq. SEQ ID No. 20

Example 6 Gene Distribution

The overall gene distribution of each ORF is summarized in Table 3. Atotal of 23 B. hyodysenteriae strains are analysed. The distribution isdetermined from the cumulative result of PCR using up to two differentprimer sets. All of the ORFS are present in 91-100% of the B.hyodysenteriae strains tested.

TABLE 3 GENE DISTRIBUTION OF THE B. hyodysenteriae VACCINE CANDIDATESANALYSED BY PCR USING A PANEL OF 23 DIFFERENT STRAINS Gene Distribution(%) NAV-H77 96 NAV-H105 91 NAV-H109 100 NAV-H114 100 NAV-H116 91NAV-H122 91 NAV-H147 100 NAV-H155 100 NAV-H161 100 NAV-H173 100

Example 7 Plasmid Extraction

Escherichia coli JM109 clones harbouring the pET-19b plasmid (Novagen)are streaked out from glycerol stock storage onto Luria-Bertani (LB)agar plates supplemented with 100 mg/l ampicillin and incubated at 37°C. for 16 h. A single colony is used to inoculate 10 ml of LB brothsupplemented with 100 mg/l ampicillin and the broth culture wasincubated at 37° C. for 12 h with shaking. The entire overnight cultureis centrifuged at 5,000×g for 10 min and the plasmid contained in thecells extracted using the QIAprep Spin Miniprep Kit (Qiagen) accordingto the manufacturer's instructions. The purified plasmid is quantifiedusing a fluorometer and the DNA concentration adjusted to 100 μg/ml bydilution with TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) buffer. Thepurified pET-19b plasmid is stored at −20° C.

Example 8 Vector Preparation

Two μg of the purified pET-19b plasmid is digested at 37° C. for 1-4 hin a total volume of 50 μl containing 50 mM Tris-HCl (pH 7.5), 100 mMNaCl, 10 mM MgCl₂, 0.1 mg/ml bovine serum albumin (BSA) and 5 U of NdeIand BamHT (Fermentas). The linearised vectors are verified byelectrophoresing 1 μl of the digestion reaction through a 1% (w/v)agarose gel in 1×TAE buffer at 90V for 1 h. The electrophoresed DNA isstained with 1 μg/ml ethidium bromide and viewed over UV light.

Linearised pET-19b vectors are purified using the UltraClean PCRClean-up Kit (Mo Bio Laboratories) according to the manufacturer'sinstructions. The purified linear vectors are quantified using theNanoDrop ND1000 spectrophotemeter and the DNA concentration adjusted to50 μg/ml by dilution with TE buffer. The linearised vectors are storedat −20° C.

Example 9 Insert Preparation—Primer Design

Primer pairs are designed to amplify as much of the coding region of thetarget gene as possible. All primers sequences include terminalrestriction enzyme recognition sites to enable cohesive-end ligation ofthe resultant amplicon into the linearised pET-19b vector. The primersequences used for cloning are shown in Table 4. The primers are testedusing Amplify 3.0 (University of Wisconsin) and the theoretical ampliconsequence is inserted into the appropriate position in the pET-19b vectorsequence. Deduced translation of the chimeric pET-19b expressioncassette is performed using Vector NTI Advance version 10 (Invitrogen)to confirm that the gene inserts would be in the correct reading frame.

TABLE 4OLIGONUCLEOTIDE PRIMERS USED IN THE CLONING OF THE B. hyodysenteriae GENESINTO THE PET-19B VECTOR. THE APPARENT MOLECULAR WEIGHT IS DETERMINEDFROM SDS-PAGE Predicted MW Apparent MW Primer of native of recombinantGene name Primer Sequence (5′-3′) protein (Da) protein (kDa) NAV- H77-F-ATTACATATGTCTCATGCTTTAGGTGTAG 27,291 26.6 H77 NdeI GACTTTATATC(SEQ ID No. 41) H77-R- AATTGGATCCATATAAATATCTTTCTTCT BamHI TCTTCGTC(SEQ ID No. 42) NAV- H105-F- AACTCATATGATACCTGCTACATCTGCG 35,447 49.9H105 NdeI AATATTG (SEQ ID No. 43) H105-R- TTTTGGATCCACACCTTTTTGAGGTATATBamHI TTAAAAC (SEQ ID No. 44) NAV- H109-F- TCTTCATATGGCTGACTTTGTTATGGGCT26,275 28.4 H109 NdeI TTGTAGGAAG (SEQ ID No. 45) H109-R-TCTAGGATCCAAATACATACCCATTTGG BamHI AAACCTATATC (SEQ ID No. 46) NAV-H114-F- TATACATATGTGCGGTAATAAAACAGAT 39,199 55.4 H114 NdeI ACTCAAACTAC(SEQ ID No. 47) H114-R- ATTAGGATCCTTTTTAAAAACTCCGCTGA BamHI ATCCATAG(SEQ ID No. 48) NAV- H116-F- TGTTCATATGAAAAGCGGTATTGAGATA 29,654 32.1H116 NdeI GGTATATTTGTTC (SEQ ID No. 49) H116-R-GGAAGGATCCTAACACCTATTTGGAAAC BamHI CTATATC (SEQ ID No. 50) NAV- H122-F-TCTTCATATGAAAAGCGGATTCGGAGTT 24,082 26.8 H122 NdeI GATTTAAC(SEQ ID No. 51) H122-R- GAATGGATCCCCTATTTGACCGCCTATAT BamHI CAAATC(SEQ ID No. 52) NAV- H147-F- TTTTCATATGTGTGCTACAACTTCTAAAA 45,175 57.9H147 NdeI GTACATC (SEQ ID No. 53) H147-R- CCTCGGATCCGCCTTTAAAGTAATAGTTTBamHI TATCATC (SEQ ID No. 54) NAV- H155-F- ATTACATATGCAATTTGATGCTAGCATAT28,879 30.3 H155 NdeI ATGCAC (SEQ ID No. 55) H155-R-TTTAGGATCCAATTTAAAGCAATTTCTAA BamHI ACTGCTAAATC (SEQ ID No. 56) NAV-H161-F- TAAACATATGGTAAGAGATAAATATTCA 32,298 33.0 H161 NdeI GAAGAG(SEQ ID No. 57) H161-R- TATTGGATCCCCTCTTTTATAGCTTATAG BamHI AAGCCTTAAC(SEQ ID No. 58) NAV- H173-F- TTTGCATATGAAAACAGGATTTGAGGTT 23,789 26.8H173 NdeI AATGTATTATTTC (SEQ ID No. 59) H173-R-AAATGGATCCCCAATCTGAGCACCTAAA BamHI TCAACACTTG (SEQ ID No. 60)

Example 10 Amplification of the Gene Inserts

All target gene inserts are amplified by PCR in a 100 μl total volumeusing Taq DNA polymerase and Pfu DNA polymerase. Briefly, theamplification mixture consists of PCR buffer (containing 1.5 mM ofMgCl₂), 2 U of Taq DNA polymerase, 0.01 U Pfu DNA polymerase, 0.2 mM ofeach dNTP, 0.5 μM of the appropriate primer pair and 1 μl of purifiedchromosomal DNA. The chromosomal DNA is prepared from the same B.hyodysenteriae strain used for genome sequencing. Cycling conditionsinvolve an initial template denaturation step of 5 min at 94° C.,followed by 30 cycles of denaturation at 94° C. for 30 s, annealing at50° C. for 15 s, and primer extension at 72° C. for 1 min. The PCRproducts are subjected to electrophoresis in 1% (w/v) agarose gels in1×TAE buffer, stained with a 1 μg/ml ethidium bromide solution andviewed over UV light. After verifying the presence of the correct sizePCR product, the PCR reaction is purified using the UltraClean PCRClean-up Kit (Mo Bio Laboratories).

Example 11 Restriction Enzyme Digestion of the Gene Inserts

Thirty μl of the purified PCR product is digested in a 50 μl totalvolume with 1 U of NdeI and 1 U of BamHI. The digested insert DNA arepurified using the UltraClean PCR Clean-up Kit (Mo Bio Laboratories).Purified digested insert DNA are quantified using the NanoDrop ND1000spectrophotemeter and the DNA concentration adjusted to 10 μg/ml bydilution with TE buffer. The purified restricted insert DNA are usedimmediately for vector ligation.

Example 12 Ligation of the Gene Inserts into the pET-19b Vector

Ligation reactions are all performed in a total volume of 20 μl. Twentyfive ng of linearised pTrcHis is incubated with an equi-molar amount ofrestricted insert at 16° C. for 16 h in 30 mM Tris-HCl (pH 7.8), 10 mMMgCl2, 10 mM DTT and 1 mM ATP containing 1 U of T4 DNA ligase(Fermentas). An identical ligation reaction containing no insert DNA isalso included as a vector re-circularisation negative control.

Example 13 Transformation of pET-19b Ligations into E. Coli JM109 Cells

Competent E. coli JM109 cells are thawed from −80° C. storage on ice andthen 50 μl of the cells are transferred into ice-cold 1.5 ml microfugetubes containing 5 μl of the overnight ligation reactions. The tubes aremixed by gently tapping the bottom of each tube on the bench and left onice for 30 min. The cells are then heat-shocked by placing the tubesinto a 42° C. waterbath for 45 s before returning the tube to ice for 2min. The transformed cells are recovered in 1 ml LB broth for 1 h at 37°C. with gentle mixing. The recovered cells are harvested at 2,500×g for5 min and the cells resuspended in 50 μl of fresh LB broth. The entire50 μl of resuspended cells are spread evenly onto a LB agar platecontaining 100 mg/l ampicillin using a sterile glass rod. Plates areincubated at 37° C. for 16 h.

Example 14 Detection of Recombinant pET-19b Constructs in E. Coli by PCR

Twelve single transformant colonies for each construct are streaked ontofresh LB agar plates containing 100 mg/l ampicillin and incubated at 37°C. for 16 h. A single colony from each transformation event isresuspended in 50 μl of TE buffer and boiled for 1 min. Two μl of boiledcells are used as template for PCR. The amplification mixture consistsof 1×PCR buffer (containing 1.5 mM of MgCl₂), 1 U of Taq DNA polymerase,0.2 mM of each dNTP, 0.5 μM of the pET-19b-F primer(5′-GGAATTGTGAGCGGATAAC-3′) and 0.5 μM of the pET-19b-R primer(5′-GCAAAAAACCCCTCAAGAC-3′). Cycling conditions involve an initialtemplate denaturation step of 5 min at 94° C., followed by 30 cycles ofdenaturation at 94° C. for 30 s, annealing at 60° C. for 15 s, and aprimer extension at 72° C. for 1 min. The PCR products are subjected toelectrophoresis in 1% (w/v) agarose gels in 1×TAE buffer, stained with a1 μg/ml ethidium bromide solution and viewed over UV light.

Example 15 Purification of Recombinant pET-19b Plasmids

An E. coli JM109 clone harbouring the successfully ligated pET-19bplasmids are inoculated into 5 ml of LB broth supplemented with 100 mg/lampicillin and the broth culture is incubated at 37° C. for 12 h withshaking. The entire overnight culture is centrifuged at 5,000×g for 10min and the plasmid contained in the cells extracted using the QIAprepSpin Miniprep Kit (Qiagen) according to the manufacturer's instructions.The purified recombinant pET-19b plasmid is stored at −20° C.

Example 16 Transformation of Recombinant pET-19b Plasmids into E. coliBL21 (DE3) Cells

Competent E. coli BL21 (DE3) cells are thawed from −80° C. storage onice and then 20 μl of the cells are transferred into ice-cold 1.5 mlmicrofuge tubes containing 1 μl of the recombinant purified pET-19bplasmid. The tubes are mixed by gently tapping the bottom of each tubeon the bench and left on ice for 30 min. The cells are then heat-shockedby placing the tubes into a 42° C. waterbath for 45 s before returningthe tube to ice for 2 min. The transformed cells are recovered in 1 mlLB broth for 1 h at 37° C. with gentle mixing. Fifty μl of the recoveredcells are spread evenly onto a LB agar plate containing 100 mg/lampicillin using a sterile glass rod. Plates are incubated at 37° C. for16 h.

Example 17 Expression of Recombinant His-Tagged Proteins

Four isolated colonies of recombinant pET-19b plasmid in E. coli BL21(DE3) is inoculated into 3 ml LB broth in a 10 ml centrifuge tubecontaining 100 mg/l ampicillin and 1 mM IPTG and incubated at 37° C. for16 h with shaking. The cells are harvested by centrifugation at 5,000×gfor 10 min at 4° C. The supernatant is discarded and each pellet isresuspended with 300 μl of 1×SDS-PAGE loading buffer (250 mM Tris-HCl pH6.0, 8% w/v SDS, 200 mM DTT, 40% v/v glycerol and 0.04% w/v bromophenolblue). After boiling the tube for 5 min, the cellular debris is pelletedby centrifugation at 10,000×g for 10 min at 4° C. The supernatant istransferred to a new tube and stored at −20° C. until analysis.

Example 18 SDS-PAGE

SDS-PAGE analysis of protein involved electrophoretic separation using adiscontinuous Tris-glycine buffer system. Ten μl of the prepared celllysate is loaded into the wells of a polyacrylamide gel. The gel iscomprised of a stacking gel (125 mM Tris-HCl ph 6.8, 4% w/v acylamide,0.15% w/v bis-acrylamide and 0.1% w/v SDS) and a separating gel (375 mMTris-HCl ph 8.8, 12% w/v acylamide, 0.31% w/v bis-acrylamide and 0.1%w/v SDS). These gels are polymerised by the addition of 0.1% (v/v) TEMEDand 0.05% (w/v) freshly prepared ammonium sulphate solution and castinto the mini-Protean dual slab cell (Bio-Rad). Samples are run at 150 Vat room temperature (RT) until the bromophenol blue dye-front reachedthe bottom of the gel. Pre-stained molecular weight standards areelectrophoresed in parallel with the samples in order to allow molecularweight estimations. After electrophoresis, the gel is immediatelystained using Coomassie Brilliant Blue G250 or subjected toelectro-transfer onto nitrocellulose membrane for Western blotting.

Example 19 Western Blot Analysis

Electrophoretic transfer of separated proteins from the SDS-PAGE gel tonitrocellulose membrane is performed using the Towbin transfer buffersystem. After electrophoresis, the gel is equilibrated in transferbuffer (25 mM Tris, 192 mM glycine, 20% v/v methanol, pH 8.3) for 15min. The proteins in the gel are transferred to nitrocellulose membraneusing the mini-Protean transblot apparatus (Bio-Rad). After assembly ofthe gel holder according to the manufacturer's instructions,electrophoretic transfer is performed at 30 V overnight at 4° C. Thefreshly transferred nitrocellulose membrane containing the separatedproteins is blocked with 10 ml of tris-buffered saline (TBS; 20 mMTris-HCl, 500 mM NaCl, pH 7.5) containing 5% (w/v) skim milk powder for1 h at RT. The membrane is washed with TBS containing 0.1% (v/v) Tween20 (TBST) and then incubated with 10 ml mouse anti-his antibody (diluted5,000-fold with TBST) for 1 h at RT. After washing three times for 5 minwith TBST, the membrane is incubated with 10 mL goat anti-mouse IgG(whole molecule)-AP diluted 5,000-fold in TBST for 1 h at RT. Themembrane is developed using the Alkaline Phosphatase Substrate Kit(Bio-Rad). The development reaction is stopped by washing the membranewith distilled water. The membrane is then dried and scanned forpresentation.

Example 20 Expression and Purification of Recombinant His-TaggedProteins

A single colony of the recombinant pET-19b plasmid in E. coli BL21 (DE3)is inoculated into 20 ml LB broth in a 50 ml centrifuge tube containing100 mg/l ampicillin and incubated at 37 C for 16 h with shaking. A 21conical flask containing 500 ml of LB broth supplemented with 100 mg/lampicillin is inoculated with 10 ml of the overnight culture andincubated at 37° C. until the optical density of the cells at 600 nm is0.5 (approximately 3-4 h). The culture is then induced by adding IPTG toa final concentration of 1 mM and the cells returned to 37° C. withshaking. After 6 h of induction, the culture is transferred to two 250ml centrifuge bottles and the bottles are centrifuged at 5,000×g for 10min at 25° C. The supernatant is discarded and each pellet isresuspended with 4 ml of Ni-NTA native lysis buffer (50 mM NaH₂PO₄, 300mM NaCl, 10 mM imidazole, pH 8.0). The resuspended cells are pooled andstored at −20° C. overnight.

The cell suspension is removed from −20° C. storage and thawed on ice.Five μl of DNAse I (10 mg/ml) is added to the thawed lysate andincubated on ice for 1 h. The lysate is centrifuged at 20,000×g for 15min at 4° C. The supernatant is transferred to a 10 ml column containinga 1 ml bed volume of Ni-NTA agarose resin (Qiagen). The recombinanthis-tagged protein is allowed to bind to the resin for 1 h at 4° C. withend-over-end mixing. The resin is then ished with 30 ml of Ni-NTA nativeish buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, pH 8.0) beforeelution with 9 ml of Ni-NTA native elution buffer (50 mM NaH₂PO₄, 300 mMNaCl, 250 mM imidazole, pH 8.0). Three 3 ml fractions of the eluate arecollected and stored at 4° C. Thirty μl of each eluate is treated with10 μl of 4× sample treatment buffer and boiled for 5 min. The samplesare subjected to SDS-PAGE and stained with Coomassie Brilliant BlueG250. The stained gel is equilibrated in distilled water for 1 h anddried between two sheets of cellulose overnight at RT.

Example 21 Dialysis and Lyophilisation of the Purified RecombinantHis-Tagged Protein

The eluted proteins are pooled and transferred into a hydrated dialysistube with a molecular weight cut-off (MWCO) of 3,500 Da. A 200 μlaliquot of the pooled eluate is taken and quantified using a commercialProtein Assay (Bio-Rad). The proteins are dialysed against 21 ofdistilled water at 4° C. with stirring. The dialysis buffer is changed 8times at 12-hourly intervals. The dialysed proteins are transferred fromthe dialysis tube into a 50 ml centrifuge tubes (40 ml maximum volume)and the tubes are placed at −80° C. overnight. Tubes are placed into afreeze-drier and lyophilised to dryness. The lyophilised proteins arethen re-hydrated with PBS to a calculated concentration of 2 mg/ml andstored at −20° C.

Example 22 Serology Using Recombinant Protein

Ten μg of purified recombinant protein is diluted in 10 ml of carbonatebuffer and 100 μl is added to each well of a 96-well microtitre plate.The protein is allowed to coat overnight at 4° C. The plate is blockedwith 150 μl of PBS-BSA (1% w/v) in each well for 1 hour at roomtemperature (RT) with mixing and then washed three times with 150 μl ofPBST (0.05% v/v). Pig sera are diluted 1:800 in 100 μl of PBST-BSA (0.1%w/v) and incubated at RT for 2 hours with mixing. Plates are washedbefore adding 100 μl of goat anti-swine IgG (whole molecule)-HRP diluted1:5,000 in PBST. After incubating for 1 hr at RT, the plates are washedand 100 μl of TMB substrate added. Colour development is allowed tooccur for 10 minutes at RT before being stopped with the addition of 50μl of 1 M sulphuric acid. The optical density of each well is read at450 nm. Pooled serum from pigs of different sources and health statuswere used in this analysis. These included pigs from high health statusherds (N1-N3), hyperimmunised pigs (M1-M3), experimentally challengedpigs (H1-H5) and recovered pigs from different herds (H6-H13).

As shown in Tables 5A and 5B, all proteins reacted strongly with serumfrom the pig hyperimmunised with B. hyodysenteriae bacteria (M1;1.504310.1417) and reacted less strongly with serum from pigshyperimmunised with B. pilosicoli (M2; 1.107510.1657) and B. innocens(M3; 1.121710.1584) indicating some level of cross-reactivity with pigsrecognising other Brachyspira spp. The proteins reacted weakest withserum taken from high-health status pigs (N1-N3; 0.3008 to 0.4488),followed by experimentally challenged pigs showing acute severe symptomsof SD (H1-H5; 0.4514 to 0.9890), and strongest with pigs which hadrecovered from SD (H6-H13; 0.8039 to 1.0053). As a whole, these resultsindicate that all these OMP are immunogenic in naturally andexperimentally infected pigs.

TABLE 5A REACTIVITY OF TOXIN PROTEINS WITH HEALTHY PIG SERUM ANDHYPERIMMUNISED PIG SERUM. ELISA Reactivity (absorbance) Protein N1 N2 N3M1 M2 M3 H77 0.3875 0.4382 0.4391 1.5461 1.0049 1.1034 H105 0.32860.4420 0.4478 1.4825 1.3311 1.1852 H109 0.3102 0.3127 0.3813 1.45611.0572 1.1465 H114 0.3786 0.3685 0.3789 1.4954 1.0146 1.0335 H116 0.37120.4488 0.4003 1.5946 1.0794 0.9309 H122 0.4407 0.3143 0.3369 1.47871.0216 1.1087 H147 0.3516 0.3492 0.3279 1.5197 1.2794 1.2972 H155 0.30080.3861 0.3764 1.4613 1.0717 1.1685 N1, N2, N3: pooled serum fromhigh-health status pigs; M1: serum from pig hyperimmunised with B.hyodysenteriae bacterin; M2: serum from pig hyperimmunised with B.pilosicoli bacterin; M3: serum from pig hyperimmunised with B. innocensbacterin.

TABLE 5B REACTIVITY OF TOXIN PROTEINS WITH NATURALLY AND EXPERIMENTALLYINFECTED PIG SERUM. ELISA Reactivity (absorbance) Protein H1 H2 H3 H4 H5H6 H7 H8 H9 H10 H11 H12 H13 H77 0.6616 0.6254 0.7151 0.7157 0.8161 0.8830.9423 0.9849 0.8030 0.9857 0.9597 0.9944 0.8550 H105 0.6664 0.72210.775 0.5747 0.8415 0.8097 0.9992 0.8891 0.8278 0.8657 0.8053 0.91450.8470 H109 0.7616 0.7699 0.6373 0.6871 0.9687 0.8703 0.8235 0.86390.9431 0.8524 0.8612 0.9485 0.9471 H114 0.7472 0.5604 0.7420 0.55130.9620 0.8422 0.9669 0.9947 0.9576 0.9885 0.9674 0.9999 0.8154 H1160.6574 0.5964 0.4580 0.5442 0.9314 0.9241 0.9110 0.8690 0.8039 0.81730.9652 0.9938 0.9128 H122 0.5198 0.5043 0.5647 0.7717 0.8122 0.89550.8049 0.9258 0.9486 0.9854 0.8141 0.9292 0.9236 H147 0.5795 0.60420.7623 0.4514 0.989 0.905 1.0002 0.8228 0.9975 0.8799 0.9884 1.00531.0031 H155 0.6238 0.6096 0.6202 0.6937 0.9357 1.0025 0.8724 0.88370.8836 0.9419 0.8043 0.8777 0.9012 H1, H2, H3, H4, H5; pooled serum fromexperimentally infected pigs showing acute severe clinical signs of SDH6, H7, H8, H9, H10, H11, H12, H13; pooled serum from commercial pigsrecovered from SD

Cloning of the various inserts into the pET-19b expression vectorproduced recombinant constructs of various sizes. Nucleotide sequencingof the pET-19b constructs verified that the expression cassette is inthe correct frame for all the constructs. The predicted translation ofthe pET-19b expression cassette indicated that all the recombinanthis-tagged proteins and the deduced amino acid sequence of the nativespirochaete proteins were identical.

Example 24 Expression and Purification of Recombinant Proteins

Expression of the selected recombinant E. coli clones is performed inmedium-scale to generate sufficient recombinant protein for vaccinationof mice. All genes cloned produce recombinant proteins possessing thehexa-histidine fusion with an apparent molecular weight similar to thepredicted molecular weight of the native protein as shown in Table 4.All recombinant proteins were highly reactive in western blotting usingthe anti-his antibody. Purification of the his-tagged recombinantproteins is by affinity chromatography under denaturing conditions.SDS-PAGE and Coomassie Blue staining of all recombinant proteins showpurification of the proteins.

Example 25 Vaccination of Pigs Using the Purified Recombinant His-TaggedProteins

Multiple purified recombinant his-tagged proteins (0.5 mg of each) werepooled in a total volume of 1 ml. Three vaccines were made:

-   -   (A) Recombinant vaccine 1 consisting of: NAV-H77, NAV-H105,        NAV-H109 and NAV-H114;    -   (B) Recombinant vaccine 2 consisting of NAV-H116, NAV-H122,        NAV-H147 and NAV-H155;    -   (C) Recombinant vaccine 3 consisting of NAV-H161 and NAV-H173.

Three groups of ten sero-negative pigs (5 weeks old) were injectedintramuscularly with 2 ml of vaccine consisting of 2 mg of the pooledantigen emulsified with a water-in-oil adjuvant. The pigs werevaccinated twice intramuscularly into the back of the neck at 5 weeks ofage and at 8 weeks of age. All pigs were bled immediately before thefirst vaccination, immediately before the second vaccination and twoweeks after the second vaccination. Serum was collected from the bloodand used to measure immune-responsiveness to the proteins contained inthe vaccines. Since four proteins are combined in each vaccine, serumfrom each group of pigs is tested against all four proteinsindividually. The ELISA was performed the same way as outlined inExample 22.

As shown in Table 6, all pigs responded strongly to the vaccinationregime given with antibody levels increasing significantly after thefirst (9.08-fold-16.53-fold increase; P<0.001) and second(2.27-fold-2.59-fold increase; P<0.001) intramuscular injections. Theseresults clearly demonstrate the immunogenicity of these OMP proteins inpigs and the effectiveness of the vaccination regime in inducing highantibody levels in the pig.

TABLE 6 IMMUNOGENICITY OF TOXIN PROTEINS IN PIGS FOLLOWING INTRAMUSCULARVACCINATION. ALL ELISA VALUES ARE EXPRESSED AS MEAN ABSORBANCE ±STANDARD DEVIATION Pre-Vacc. Pre-Boost Post-vaccination (2 weeks)Protein ELISA value ELISA Value Fold increase ELISA value Fold increaseH77 0.0671 ± 0.0295 1.1087 ± 0.2037 16.53 2.7387 ± 0.2990 2.47 H1050.0663 ± 0.0257 1.0746 ± 0.2087 16.20 2.6299 ± 0.2854 2.45 H109 0.0822 ±0.0235 1.0443 ± 0.1932 12.70 2.7071 ± 0.2785 2.59 H114 0.0661 ± 0.02801.0480 ± 0.2105 15.85 2.5951 ± 0.2955 2.48 H116 0.1096 ± 0.0244 0.9952 ±0.1455 9.08 2.2555 ± 0.2482 2.27 H122 0.0904 ± 0.0327 1.0498 ± 0.268611.62 2.6590 ± 0.2354 2.53 H147 0.0682 ± 0.0354 1.0654 ± 0.1386 15.632.6027 ± 0.2085 2.44 H155 0.0831 ± 0.0298 1.0407 ± 0.2497 12.52 2.6112 ±0.3649 2.51

Example 26 Protection of Pigs by Vaccination with Purified RecombinantHis-Tagged Proteins

Multiple purified recombinant his-tagged proteins (0.5 mg of each) arepooled in a total volume of 1 ml. Three vaccines are made:

-   -   a) Recombinant vaccine 1 consisting of: NAV-H77, NAV-H105,        NAV-H109 and NAV-H114;    -   b) Recombinant vaccine 2 consisting of: NAV-H116, NAV-H122,        NAV-H147 and NAV-H155;    -   c) Recombinant vaccine 3 consisting of NAV-H161 and NAV-H173.

Four groups of ten sero-negative pigs (5 weeks old) are injectedintramuscularly with 2 ml of vaccine consisting of 2 mg of the pooledantigen emulsified with a water-in-oil adjuvant. A fifth group of tensero-negative pigs is used as negative controls and are sham vaccinatedwith PBS emulsified in the adjuvant. All the pigs are vaccinated twiceintramuscularly into the back of the neck at 5 weeks of age and at 8weeks of age. All pigs are challenged with 100 ml of an active Bhyodysenteriae culture (˜10⁹ cells/ml) by gastric gavage at ten weeks ofage, and pigs are observed daily for clinical signs of swine dysenteryduring the experiment (up to six weeks post-challenge) and atpost-mortem examination. Twice weekly, all pigs are rectally swabbed forselective bacteriological culture.

The invention claimed is:
 1. An isolated polynucleotide that encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:
 8. 2. Theisolated polynucleotide of claim 1, wherein the isolated polynucleotidecomprises the nucleotide sequence of SEQ ID NO:
 7. 3. A plasmidcomprising the polynucleotide of claim
 1. 4. The plasmid of claim 3,wherein said plasmid is an expression vector.
 5. A cell containing theplasmid of claim
 3. 6. An isolated protein comprising the amino acidsequence of SEQ ID NO:
 8. 7. An immunogenic composition comprising: (a)a plasmid comprising a polynucleotide that encodes the amino acidsequence of SEQ ID NO:8, (b) a cell containing the plasmid comprising apolynucleotide that encodes the amino acid sequence of SEQ ID NO:8, or(c) an isolated protein comprising the amino acid sequence of SEQ ID NO:8.
 8. A method of diagnosing Brachyspira infection comprising: (a)providing a sample from an animal suspected of being infected withBrachyspira; (b) contacting the sample with one or more isolatedpolypeptides comprising an amino acid sequence of SEQ ID NO: 8; (c)incubating the sample and polypeptide under conditions which allow forthe formation of antibody-antigen complexes; and (d) determining whetheran antibody-antigen complex with one or more polypeptides is formed,wherein the formation of an antibody-antigen complex indicates theanimal is infected with Brachyspira.
 9. The method of claim 8, whereinthe Brachyspira infection is infection with Brachyspira hyodysenteriae.10. A kit for diagnosing Brachyspira infection comprising one or moreisolated polypeptides comprising an amino acid sequence of SEQ ID NO: 8.